Flow cell devices and use thereof

Abstract

The present disclosure provides flow cell devices, systems, and methods for facilitating and performing DNA sequencing assays with reduced system complexity and cost, significant COGS savings, and reduced levels of contamination. In some embodiments, the flow cell devices and systems may include: a carrier including one or more substrates; one or more channels defined by the one or more substrates and configured to allow fluid flow therethrough; an inlet in fluid connection with the one or more channels, the inlet including an open landing area in a substrate; and an outlet.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480017304.0 (22) Application Date 2024.01.08 (30) Priority Data 63 / 479,158 2023.01.09 US 63 / 502,896 2023.05.17 US (85) PCT International Application Entering National Phase Date 2025.09.05 (86) PCT International Application Application Data PCT / US2024 / 010760 2024.01.08 (87) PCT International Application Publication Data WO2024 / 151556 EN 2024.07.18 (71) Applicant: Element Biosciences, Inc. Address: California, USA (72) Inventors: B.K. Lin, Yu Yingxian, Lu Jingzhi, M.C. Ray, Xing Siyuan, Zhang Qiting, O. Kandan, J. Ye, D. Hastings, M.T. Dangro, Wu Jueyu (74) Patent Agency: King & Wood Mallesons, Beijing 11256 Patent Attorneys: Chen Wenping, Wang Beinan (51) Int.Cl. B01L 3 / 00 (2006.01) C12Q 1 / 6874 (2006.01) G01N 33 / 49 (2006.01) G01N 21 / 05 (2006.01) G01N 30 / 88 (2006.01) (54) Title of Invention: Flow Cell Apparatus and Its Use (57) Abstract This disclosure provides flow cell apparatus, systems, and methods for facilitating and performing DNA sequencing analyses, which feature reduced system complexity and cost, significant COGS savings, and reduced contamination levels. The flow cell apparatus and system may include: a carrier comprising one or more substrates; one or more channels defined by the one or more substrates and configured to allow fluid flow through them; an inlet fluidly connected to the one or more channels, the inlet comprising an open landing area in a substrate; and an outlet. Claims 22 pages, Description 105 pages, Drawings 79 pages, CN 121057626 A 2025.12.02 CN 1 21 05 76 26 A 1. A flow cell apparatus comprising: (a) a carrier including one or more substrates, wherein the one or more substrates include an inlet and an outlet, wherein the inlet includes an open landing area; and (b) one or more channels defined by the one or more substrates, wherein the one or more channels are fluidly connected to the inlet and the outlet, wherein the one or more channels are configured to allow fluid or an air gap between the fluid and another fluid to flow through the one or more channels.2. The flow cell apparatus of claim 1, wherein the open landing area is at least partially covered with a surface coating. 3. The flow cell apparatus of claim 1, wherein the one or more channels extend from the inlet to the outlet. 4. The flow cell apparatus of claim 1, wherein the one or more channels extend along a first direction between the inlet and the outlet. 5. The flow cell apparatus of claim 1, wherein the one or more channels are configured to allow air gap flow through the one or more channels, wherein the fluid contains a first reagent, and the other fluid contains a second reagent. 6. The flow cell apparatus of claim 5, wherein the one or more channels are configured to allow air gap flow through the one or more channels during a DNA sequencing run. 7. The flow cell apparatus of claim 1, wherein the one or more channels are configured to allow air gap flow through the one or more channels from the inlet. 8. The flow cell apparatus of claim 6, wherein the one or more channels are configured to allow air gap flow through the one or more channels to facilitate reduced contamination of the first reagent to the second reagent during the DNA sequencing run. 9. The flow cell apparatus of claim 6, wherein the one or more channels are configured to allow air gap flow through the one or more channels to reduce the minimum amount of the first reagent, the second reagent, or the washing reagent used in the DNA sequencing run. 10. The flow cell apparatus of claim 1, wherein the one or more channels comprise one or more surfaces. 11. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an inner surface. 12. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an outer surface. 13. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an inner top surface, an inner bottom surface, or both. 14. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an outer top surface, an outer bottom surface, or both. 15. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise a planar surface. 16. The flow cell apparatus of claim 10, wherein the one or more surfaces are passivated. 17. The flow cell apparatus of claim 10, wherein the one or more surfaces are passivated with a coating that immobilizes the surface to capture primers, nucleic acid template molecules, or both, to capture polynucleotides. 18. The flow cell apparatus of claim 17, wherein the one or more surfaces comprise the polynucleotide coupled thereto. Claims 1 / 22 pages 2 CN 121057626 A19. The flow cell apparatus of claim 10, wherein the air gap is configured to remove moisture or liquid from at least a portion of the one or more surfaces of the one or more channels. 20. The flow cell apparatus of claim 10, wherein the air gap does not impair the chemical function of the one or more surfaces. 21. The flow cell apparatus of claim 17, wherein the coating of the one or more surfaces comprises at least one hydrophilic polymer coating. 22. The flow cell apparatus of claim 17, wherein the coating of the one or more surfaces comprises a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating. 23. The flow cell apparatus of claim 10, wherein the one or more surfaces comprises at least one discrete region comprising a plurality of cloned amplified sample nucleic acid molecules annealed to a plurality of attached oligonucleotide molecules. 24. The flow cell system apparatus of claim 21, wherein the water contact angle of the at least one hydrophilic polymer coating does not exceed about 50 degrees. 25. The flow cell apparatus of claim 23, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a tandem polymer, the tandem polymer being annealed to at least one of the plurality of attached oligonucleotide molecules. 26. The flow cell apparatus of claim 21, wherein the at least one hydrophilic polymer coating comprises polyethylene glycol (PEG). 27. The flow cell apparatus of claim 21, wherein the one or more surfaces further comprise a second hydrophilic polymer coating. 28. The flow cell apparatus of claim 21, wherein the at least one hydrophilic polymer coating comprises a branched hydrophilic polymer. 29. The flow cell apparatus of claim 28, wherein the branched hydrophilic polymer comprises at least eight branches. 30. The flow cell apparatus of claim 23, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule, the single-stranded multimeric nucleic acid molecule comprising a repeating sequence of regularly occurring monomeric units. 31. The flow cell apparatus of claim 30, wherein the single-stranded multimeric nucleic acid molecule has a length of at least 10 kilobases. 32. The flow cell apparatus of claim 30, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules further comprises a double-stranded monomer copy of the regularly occurring monomer unit. 33. The flow cell apparatus of claim 22, wherein the plurality of oligonucleotide molecules are present at various locations on the one or more surfaces with a substantially uniform surface density.34. The flow cell apparatus of claim 22, wherein the plurality of oligonucleotide molecules are present at a first location on the one or more surfaces at a local surface density of at least 100,000 molecules / μm², and at a second location on the one or more surfaces at a second local surface density. 35. The flow cell apparatus of claim 10, wherein the coating comprises: (a) a first layer comprising monolayer polymer molecules tethered to the surface of one or more substrates; (b) a second layer comprising a second monolayer polymer molecules tethered to the polymer molecules of the first layer; and (c) a third layer comprising a third monolayer polymer molecules tethered to the polymer molecules of the second layer, wherein at least one of the first layer, the second layer, or the third layer comprises branched polymer molecules. 36. The flow cell apparatus of claim 35, wherein the third layer further comprises oligonucleotides tethered to the polymer molecules of the third layer. 37. The flow cell apparatus of claim 36, wherein the oligonucleotides tethered to the polymer molecules of the third layer are distributed throughout the third layer at multiple depths. 38. The flow cell apparatus of claim 17, wherein the coating further comprises: (a) a fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer, and (b) a fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer. 39. The flow cell apparatus of claim 38, wherein the polymer molecules of the fifth layer further comprise oligonucleotides tethered to the polymer molecules of the fifth layer. 40. The flow cell apparatus of claim 39, wherein the oligonucleotides tethered to the polymer molecules of the fifth layer are distributed throughout the fifth layer at multiple depths. 41. The flow cell apparatus of claim 21, wherein the at least one hydrophilic polymer coating comprises: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran.42. The flow cell apparatus of claim 23, wherein when the plurality of clone-amplified sample nucleic acid molecules or their complementary sequences are labeled with cyanine dye-3, the ratio of the fluorescence intensity of the cyanine dye-3-labeled sample nucleic acid molecules or their complementary sequences shown in the images of the one or more surfaces to the fluorescence intensity of the non-specific cyanine dye-3 adsorption background (B gap) is at least 3:1. 43. The flow cell apparatus of claim 42, wherein the ratio of the fluorescence intensity of the cyanine dye-3-labeled sample nucleic acid molecules or their complementary sequences shown in the images of the one or more surfaces to the fluorescence intensity of the combination of the non-specific cyanine dye-3 adsorption background and the non-specific amplification background (B gap + B endoplasm) is at least 3:1. 44. The flow cell apparatus of claim 23, wherein when the plurality of clone-amplified sample nucleic acid molecules or their complementary sequences are labeled with cyanine dye-3, the ratio of the fluorescence intensity of the cyanine dye-3-labeled sample nucleic acid molecules or their complementary sequences, as shown in the images of the one or more surfaces, to the fluorescence intensity of the nonspecific dye adsorption background (B gap) is at least 5:1. 45. The flow cell apparatus of claim 44, wherein the ratio of the fluorescence intensity of the cyanine dye-3-labeled sample nucleic acid molecules or their complementary sequences, as shown in the images of the one or more surfaces, to the fluorescence intensity of the combination of the nonspecific cyanine dye-3 adsorption background and the nonspecific amplification background (B gap + B endoplasm) is at least 5:1. 46. ​​The flow cell apparatus of claim 23, wherein, when the plurality of cloned amplified sample nucleic acid molecules or their complementary sequences are labeled with cyanine dye-3, and when the fluorescence images of the one or more surfaces are acquired under non-signal saturation conditions using an inverted microscope equipped with a 20× objective lens, NA=0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for cyanine dye-3 emission, and a camera, the fluorescence images exhibit a contrast-to-noise ratio (CNR) of at least 20. 47. The flow cell apparatus of claim 22, wherein the plurality of oligonucleotide molecules are present at a surface density of at least 1,000 molecules / m². 48. The flow cell apparatus of claim 10, wherein the first reagent is configured to wet the one or more surfaces of the one or more channels. 49. The flow cell apparatus of claim 19, wherein the second reagent is configured to rewet the surfaces after at least a portion of moisture or liquid has been removed from the one or more surfaces of the one or more channels.50. The flow cell apparatus of claim 1, wherein the air gap comprises air. 51. The flow cell apparatus of claim 1, wherein the air gap comprises dry air. 52. The flow cell apparatus of claim 1, wherein the air gap comprises one or more inert gases. 53. The flow cell apparatus of claim 1, wherein the air gap comprises one or more active gases. 54. The flow cell apparatus of claim 5, wherein the first reagent or the second reagent comprises a liquid. 55. The flow cell apparatus of claim 5, wherein the first reagent or the second reagent does not contain bubbles larger than a predetermined size. 56. The flow cell apparatus of claim 17, wherein the coating comprises a liquid-repellent coating. 57. The flow cell apparatus of claim 17, wherein the coating comprises a superhydrophobic coating. 58. The flow cell apparatus of claim 17, wherein the coating comprises a smooth liquid-filled porous surface (SLIPS). 59. The flow cell apparatus of claim 17, wherein the coating comprises a smooth superhydrophobic covalently attached liquid (SOCAL) coating. 60. The flow cell device of claim 17, wherein the coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates. 61. The flow cell device of claim 17, wherein the coating is formed by impregnating a lubricant into one or more porous surfaces. 62. The flow cell device of claim 61, wherein the lubricant comprises a liquid with a surface energy below about 20 mJ / m². 63. The flow cell device of claim 61, wherein the lubricant comprises silicone oil. 64. The flow cell device of claim 17, wherein the surface energy of the coating is below about 20 mJ / m². 65. The flow cell device of claim 17, wherein the coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers. 66. The flow cell device of claim 65, wherein the one or more brine monomers comprise dimethyldimethoxysilane. 67. The flow cell device of claim 1, wherein the open landing area is fluidly connected to the one or more channels. 68. The flow cell apparatus of claim 1, wherein the open landing region is fluidly connected to one of the one or more channels. 69. The flow cell apparatus of claim 1, wherein the open landing region is fluidly connected to two or more of the one or more channels. 70. The flow cell apparatus of claim 1, wherein the open landing region is located on the bottom substrate of the one or more substrates.71. The flow cell device of claim 1, wherein the inlet comprises a hole in a top substrate of the one or more substrates. 72. The flow cell device of claim 71, wherein the hole in the top substrate is positioned above at least a portion of the open landing region. 73. The flow cell device of claim 72, wherein the flow cell device is configured to allow a dispenser to openly dispense one or more reagents through the hole to the open landing region. 74. The flow cell device of claim 73, wherein the dispenser is configured to openly dispense the one or more reagents from the tip of the dispenser to the open landing region. 75. The flow cell device of claim 74, wherein the dispenser is configured to openly dispense the one or more reagents from the tip of the dispenser to the open landing region without requiring a conduit between the dispenser and the open landing region. 76. The flow cell device of claim 75, wherein at least a portion of the tip of the dispenser contacts the open landing region. 77. The flow cell device of claim 75, wherein the tip of the dispenser does not contact the open landing region. 78. The flow cell apparatus of claim 1, further comprising a cleaning outlet in one or more substrates. 79. The flow cell apparatus of claim 78, wherein the cleaning outlet is fluidly connected to the inlet. 80. The flow cell apparatus of claim 79, wherein the cleaning outlet is fluidly connected to the open landing area. 81. The flow cell apparatus of claim 80, wherein the cleaning outlet is located below the open landing area. 82. The flow cell apparatus of claim 78, wherein the cleaning outlet is located in a top substrate or a bottom substrate of one or more substrates. 83. The flow cell apparatus of claim 78, wherein the cleaning outlet comprises a side port on one or more substrates, wherein the side port: (a) extends at least along a direction perpendicular to or substantially perpendicular to the x-direction; (b) extends at least along a direction perpendicular to or substantially perpendicular to the y-direction; (c) extends at least along a direction perpendicular to or substantially perpendicular to the z-direction; (d) extends at least along a direction inclined to the x-direction; (e) extends at least along a direction inclined to the y-direction; or (f) extends at least along a direction inclined to the z-direction. Claims 5 / 22, page 6, CN 121057626 A 84. The flow cell apparatus of claim 78, wherein the cleaning outlet is configured to be connected to a first pump or a second pump. 85. The flow cell apparatus of claim 1, wherein the one or more channels comprise one or more microfluidic channels.86. The flow cell apparatus of claim 10, wherein the one or more surfaces are coated with fluorescent beads, the fluorescent beads being chemically fixed to the one or more surfaces. 87. The flow cell apparatus of claim 86, wherein the fluorescent beads are covalently attached to the one or more surfaces. 88. The flow cell apparatus of claim 13, wherein the gap between the inner top surface and the inner bottom surface is approximately 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. 89. The flow cell apparatus of claim 1, wherein the height of the one or more channels is approximately 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. 90. The flow cell apparatus of claim 18, wherein the polynucleotides captured thereon are configured for imaging in a sequencing cycle. 91. The flow cell apparatus of claim 1, wherein the one or more substrates comprise a top substrate and a bottom substrate. 92. The flow cell apparatus of claim 91, wherein the one or more channels are defined between the top substrate and the bottom substrate. 93. The flow cell apparatus of claim 91, wherein the one or more channels are at least partially defined in the top surface of the bottom substrate. 94. The flow cell apparatus of claim 91, wherein the one or more channels are at least partially defined in the bottom surface of the top substrate. 95. The flow cell apparatus of claim 91, wherein the one or more substrates further comprise an intermediate substrate. 96. The flow cell apparatus of claim 95, wherein the one or more channels are at least partially defined in the intermediate substrate. 97. The flow cell apparatus of claim 1, wherein the one or more substrates comprise glass or plastic. 98. The flow cell apparatus of claim 1, wherein at least a portion of the carrier is transparent. 99. The flow cell apparatus of claim 1, wherein at least a portion of the one or more substrates is transparent. 100. The flow cell apparatus of claim 1, wherein the carrier is solid. 101. The flow cell apparatus of claim 1, wherein the one or more channels comprise 1, 2, 3, 4, 5, 6, 7, or 8 channels. 102. The flow cell apparatus of claim 1, wherein the one or more channels comprise 2, 4, 6, 8, or 10 channels. 103. The flow cell apparatus of claim 1, wherein the lane length of each of the one or more channels is less than about 70 mm, 75 mm, 80 mm, or 90 mm.104. The flow cell apparatus of claim 1, wherein the lane width of each of the one or more channels is less than about 10 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, or 3 mm. (Claims 6 / 22, page 7, CN 121057626 A) 105. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising a smooth coating. 106. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising a liquid-repellent coating. 107. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising a superhydrophobic coating. 108. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising a smooth liquid injection porous surface (SLIPS). 109. The flow cell apparatus of claim 2, wherein at least a portion of the open landing region is covered with a second surface coating comprising a smooth, superhydrophobic, covalently attached liquid (SOCAL) coating. 110. The flow cell apparatus of claim 2, wherein at least a portion of the open landing region is covered with a second surface coating comprising a liquid-like polymer brush surface covalently attached to the one or more substrates. 111. The flow cell apparatus of claim 2, wherein at least a portion of the open landing region is covered with a second surface coating comprising impregnating a lubricant in a porous surface to produce a second surface coating with a surface energy below about 20 mJ / m². 112. The flow cell apparatus of claim 2, wherein at least a portion of the open landing region is covered with a second surface coating comprising an acid-catalyzed graft polycondensation of one or more brine monomers. 113. The flow cell apparatus of claim 112, wherein the one or more brine monomers comprise dimethyldimethoxysilane. 114. The flow cell apparatus of claim 5, wherein the flow cell apparatus is configured to allow at least a portion of the first reagent to be cleaned from at least a portion of the one or more channels during a DNA sequencing run. 115. The flow cell apparatus of claim 5, wherein the flow cell apparatus is configured to allow at least a portion of the first reagent to remain in the one or more channels. 116. The flow cell apparatus of claim 5, wherein the first reagent and the second reagent are different. 117. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies the...118. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than half of the corresponding volume or length of each of the one or more channels. 119. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than 60% of the corresponding volume or length of each of the one or more channels. 120. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than 70% of the corresponding volume or length of each of the one or more channels. 121. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than 80% of the corresponding volume or length of each of the one or more channels. 122. The flow cell apparatus of claim 5, wherein the clean outlet is configured to allow residual amounts of the first reagent on the open landing area to flow through the clean outlet. 123. The flow cell apparatus of claim 122, wherein the residual amount of the first reagent on the open landing area comprises a meniscus of the first reagent. Claims 7 / 22 Page 8 CN 121057626 A 124. The flow cell device of claim 91, further comprising one or more seals positioned on the one or more substrates. 125. The flow cell device of claim 124, wherein a first portion of the channel in the one or more channels includes a first z-position, and a second portion of the channel includes a second z-position different from the first z-position. 126. The flow cell device of claim 125, wherein the first portion of the channel includes one or more first imaging surfaces. 127. The flow cell device of claim 125, wherein the second portion of the channel includes one or more second imaging surfaces. 128. The flow cell device of claim 125, wherein the top substrate or the bottom substrate includes one or more substrate layers. 129. The flow cell device of claim 125, wherein the top substrate includes a first thickness above the first portion of the channel and a second thickness around the second portion of the channel. 130. The flow cell device of claim 129, wherein the second thickness is greater than the first thickness. 131. The flow cell apparatus of claim 129, wherein the second thickness is 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the first thickness. 132. The flow cell apparatus of claim 125, wherein the bottom substrate includes the channel of the first...A third thickness above a portion and a fourth thickness above the second portion of the channel. 133. The flow pool device of claim 132, wherein the fourth thickness is greater than the third thickness. 134. The flow pool device of claim 132, wherein the fourth thickness is 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the third thickness. 135. The flow pool device of claim 124, wherein the one or more seals comprise one or more mechanical seals. 136. The flow pool device of claim 124, wherein the one or more seals comprise one or more gaskets. 137. The flow pool device of claim 79, wherein the cleaning outlet is configured to remove fluid from the one or more channels. 138. The flow pool device of claim 79, wherein the cleaning outlet is in sealed fluid connection to a pump or vacuum device. 139. The flow pool device of claim 79, wherein the cleaning outlet is configured to direct fluid or gas to the one or more channels. 140. The flow cell device of claim 91, wherein the bottom substrate comprises glass, plastic, or both. 141. The flow cell device of claim 125, wherein the one or more seals comprises a first seal having a thickness along the z-direction equivalent to the thickness of the top substrate in the second portion. 142. The flow cell device of claim 125, wherein the one or more seals comprises a second seal having a thickness along the z-direction equivalent to the thickness of the bottom substrate in the second portion. 143. The flow cell device of claim 142, wherein the thickness of the second seal along the z-direction is greater than the thickness of the bottom substrate in the first portion. 144. The flow cell device of claim 124, wherein the flow cell device further comprises a frame covering at least a portion of the one or more substrates. Claims 8 / 22 Page 9 CN 121057626 A 145. The flow cell device of claim 144, wherein the frame is mechanically secured to the one or more seals. 146. The flow cell device of claim 144, wherein the frame comprises plastic. 147. The flow cell apparatus of claim 125, wherein the one or more seals interface with a manifold or connector to allow sealed fluid communication between the manifold or connector and the one or more channels. 148. The flow cell apparatus of claim 147, wherein the manifold or connector comprises one or more fluid passages.149. The flow pool apparatus of claim 148, wherein the one or more fluid passages are in fluid communication with the one or more channels. 150. The flow pool apparatus of claim 148, wherein the one or more fluid passages are in fluid communication with the open landing area. 151. The flow pool apparatus of claim 148, wherein the manifold or the connector is configured to be in sealed fluid communication with the one or more channels by applying a pressure thereon that satisfies a predetermined threshold. 152. The flow pool apparatus of claim 151, wherein the one or more fluid passages extend along a y-axis, and wherein the pressure is applied along the y-axis. 153. The flow pool apparatus of claim 151, wherein the one or more fluid passages extend along an x-axis, and wherein the pressure is applied along the x-axis. 154. The flow pool apparatus of claim 148, wherein the manifold or the connector includes a coupling interface that directly contacts an end of the flow pool apparatus. 155. The flow pool apparatus of claim 148, wherein the manifold or the connector includes a coupling interface that contacts an end of the flow pool apparatus with the one or more seals therebetween. 156. The flow cell device of claim 148, wherein the manifold or the connector includes a mating interface that contacts an end of the flow cell device with an adhesive therebetween. 157. The flow cell device of claim 148, wherein the manifold or the connector includes an open region located at an end of one or more of the fluid passages. 158. The flow cell device of claim 152, wherein the open region is fluidly connected to the open landing region. 159. The flow cell device of claim 148, further comprising one or more reference features configured to position the flow cell device relative to the manifold or the connector, the stage, or the sequencing system. 160. The flow cell device of claim 159, wherein the one or more reference features include at least one alignment feature positioned at a center point along the x-axis. 161. The flow cell device of claim 159, wherein the one or more reference features include at least one alignment feature positioned along the y-axis at or near an end of one or more substrates. 162. The flow cell apparatus of claim 159, wherein the one or more reference features include a cavity extending through the one or more substrates and configured to be coupled to a pin. 163. The flow cell apparatus of claim 159, wherein the one or more reference features include a groove, wherein...The groove extends through the one or more substrates and is configured to engage with a pin. Claims 9 / 22, page 10, CN 121057626 A 164. The flow cell device of claim 159, wherein the manifold or the connector includes a top portion or a bottom portion extending beyond the one or more substrates along the z-axis and covering at least a portion of the one or more substrates in the x-y plane. 165. The flow cell device of claim 164, wherein the top portion or the bottom portion is located at the first portion, the second portion, or both of the one or more channels. 166. The flow cell device of claim 164, wherein the top portion or the bottom portion includes one or more alignment features configured to align the top portion or the bottom portion with the flow cell device. 167. The flow cell device of claim 164, wherein the top portion or the bottom portion includes one or more alignment features configured to align the top portion or the bottom portion with the flow cell device along the z-axis or along the y-axis. 168. The flow cell device of claim 148, further comprising one or more tubes, the one or more tubes interfacing with the manifold or the connector and the flow cell device. 169. The flow cell device of claim 168, wherein each of the one or more tubes comprises a wall surrounding a lumen. 170. The flow cell device of claim 169, wherein the lumen is in fluid communication with the one or more channels of the flow cell device and the one or more fluid passages of the manifold or the connector. 171. The flow cell device of claim 170, wherein at least a portion of the one or more tubes is embedded in the one or more substrates. 172. The flow cell device of claim 171, wherein each of the one or more tubes is coupled to the manifold or the connector, thereby enabling fluid communication therebetween. 173. The flow cell device of claim 125, wherein the one or more seals comprise a protective sleeve seal covering at least a portion of the flow cell device in the x-y plane and covering one end of the flow cell device in the x-z plane. 174. The flow cell apparatus of claim 125, wherein the one or more seals comprise a flexible material that deforms under pressure satisfying a predetermined threshold. 175. The flow cell apparatus of claim 125, wherein the one or more seals comprise an L-shaped seal extending along the z-axis and the y-axis.176. The flow cell device of claim 175, wherein the L-shaped seal extends along the y-axis and into a corresponding channel in one or more of the channels. 177. The flow cell device of claim 176, wherein pressure or force is applied to the L-shaped seal along the y-axis to seal fluid communication between the flow cell device and the manifold. 178. The flow cell device of claim 148, wherein the one or more seals are configured to interface with the manifold or connector, thereby allowing sealed fluid communication between the flow cell device and the manifold. 179. The flow cell device of claim 125, wherein the one or more seals comprise a membrane seal covering at least a portion of the flow cell device and at least a portion of the manifold or connector, thereby sealing fluid communication therebetween. 180. The flow cell device of claim 179, wherein the membrane seal comprises a flat washer placed on top of the top surface of the top substrate, a flat washer placed below the bottom surface of the bottom substrate, or both. Claims 10 / 22 Page 11 CN 121057626 A 181. The flow cell device of claim 179, wherein the membrane seal extends in the x-y plane. 182. The flow cell device of claim 148, wherein the manifold or connector includes a finger-shaped notch region located between two channels of the one or more channels of the flow cell device. 183. The flow cell device of claim 182, wherein the manifold or connector includes a seal placed in the finger-shaped notch region and configured to seal fluid communication between the two channels. 184. The flow cell device of claim 148, wherein the manifold or connector includes a fluid passage having an outlet exiting the manifold in a plane orthogonal to the y-axis, the x-axis, or the z-axis. 185. The flow cell device of claim 125, wherein the top substrate or the bottom substrate includes one or more inclined ends. 186. The flow cell device of claim 185, wherein the tip of one of the one or more inclined ends presses against the one or more seals. 187. The flow cell device of claim 186, wherein each of the one or more inclined ends interfaces with an inclined manifold or connector. 188. The flow cell device of claim 187, wherein the one or more inclined ends include a first acute ramp angle relative to the y-axis. 189. The flow cell device of claim 188, wherein the inclined manifold or connector includes a second acute ramp angle relative to the y-axis.190. The flow cell device of claim 189, wherein the first acute ramp angle is different from the second acute ramp angle. 191. The flow cell device of claim 190, wherein the first acute ramp angle is the same as the second acute ramp angle. 192. The flow cell device of claim 191, wherein the inclined manifold or connector includes a ramp complementary to the inclined end of the flow cell device. 193. The flow cell device of claim 192, wherein the one or more seals include a diagonal washer having a fluid passage extending in the y-z plane. 194. The flow cell device of claim 193, wherein the diagonal washer, the manifold, or the connector interfaces with an end of the top substrate and a top surface of the bottom substrate. 195. The flow cell device of claim 193, wherein the diagonal washer, the manifold, or the connector interfaces with an end of the bottom substrate and a top inner surface of the top substrate. 196. The flow cell device of claim 193, wherein the diagonal gasket manifold or the connector allows sealed fluid communication from the fluid passage to the one or more channels when the force or pressure includes a y-axis component satisfying a first threshold and a z-axis component satisfying a second threshold. 197. The flow cell device of claim 148, wherein the top substrate and the bottom substrate are offset from each other at least laterally along the y-axis. 198. The flow cell device of claim 148, wherein at least a portion of the manifold or the connector is fixedly attached to the bottom inner surface of the bottom substrate. 199. The flow cell device of claim 148, further comprising an intermediary layer configured to define the one or more channels between the top substrate and the bottom substrate. Claims 11 / 22 pages 12 CN 121057626 A 200. The flow cell device of claim 148, wherein the top substrate and the bottom substrate are not directly fixedly attached to each other. 201. The flow cell apparatus of claim 148, wherein at least a portion of the manifold or the connector is fixedly attached to the top inner surface of the top substrate. 202. The flow cell apparatus of claim 196, wherein the diagonal washer, the manifold, or the connector's fluid passage extends at least along the y-axis. 203. The flow cell apparatus of claim 148, wherein the manifold or the connector further comprises an open well leading to a second open landing region, and wherein the second open landing region is configured to receive reagent from a dispensing tip. 204. The flow cell apparatus of claim 203, wherein the open well of the manifold or the connector is in fluid communication with the one or more channels.205. The flow cell device of claim 204, wherein the second open landing area of ​​the manifold or the connector is in fluid communication with the inlet of the one or more channels. 206. The flow cell device of claim 125, wherein the one or more seals comprise a thermoplastic connector and a thermoplastic seal mounted on the thermoplastic connector. 207. The flow cell device of claim 206, wherein the thermoplastic seal is deformable under pressure changes, temperature changes, or both. 208. The flow cell device of claim 207, wherein the thermoplastic seal comprises one or more materials different from one or more materials of the thermoplastic connector. 209. The flow cell device of claim 125, wherein the one or more seals comprise a first connector having a top portion that is slidable on a top surface of the top substrate. 210. The flow cell device of claim 209, wherein the one or more seals comprise a second connector having a bottom portion that is slidable on a bottom surface of the bottom substrate. 211. The flow cell device of claim 210, wherein the top portion is connected to a first side portion of the first connector, the first side portion being configured to interface with an end of the flow cell device in the x-z plane. 212. The flow cell device of claim 210, wherein the bottom portion is connected to a second side portion of the second connector, the second side portion being configured to interface with an end of the flow cell device in the x-z plane. 213. The flow cell device of claim 212, wherein a pressure or force satisfying a predetermined threshold on the first side portion and the second side portion is configured to cause the first connector and the second connector to slide relative to the flow cell device under deformation, thereby enabling the one or more channels to be in sealing communication with a fluid passage defined between the top connector and the bottom connector. 214. The flow cell device of claim 125, wherein the inlet includes a port that opens at the bottom surface of the bottom substrate. 215. The flow cell device of claim 214, wherein the port is in fluid communication with the one or more channels of the connector and the fluid passage. 216. The flow cell apparatus of claim 125, wherein the one or more seals comprise a semi-rigid or deformable material that deforms under pressure or force. 217. The flow cell apparatus of claim 216, wherein the semi-rigid or deformable material is configured to return to its shape prior to deformation when the pressure or force is removed. Claims 12 / 22 pages 13 CN121057626 A 218. The flow cell device of claim 125, wherein the one or more seals comprise a gasket, a second connector, a second manifold or a component thereof, or a combination thereof. 219. The flow cell device of claim 218, further comprising a force application mechanism controlled by computer-readable instructions executable on a computer processor. 220. The flow cell device of claim 219, wherein the second manifold, the second connector, or the one or more seals are connected to the force application mechanism, thereby allowing connection to or disconnection from the flow cell device. 221. A flow cell system comprising: (a) the flow cell device of any one of claims 1 to 220; (b) a fluid control device. 222. The flow cell system of claim 221, wherein the fluid control device comprises a first pump, a second pump, or both. 223. The flow cell system of claim 221, wherein the fluid control device comprises: (a) a third pump connected to the outlet of the flow cell device; and (b) a dispenser configured to openly dispense the one or more reagents to the inlet of the flow cell device. 224. The flow cell system of claim 221, wherein the fluid control device comprises: (a) a fourth pump fluidly connected to the clean outlet of the flow cell device; (b) a fifth pump fluidly connected to the outlet of the flow cell device; and (c) a dispenser configured to openly dispense the one or more reagents to the inlet of the flow cell device. 225. The flow cell system of any one of claims 221 to 224, wherein the first pump or the second pump is configured to introduce the air gap through the inlet and allow at least partial flow through the one or more channels. 226. The flow cell system of any one of claims 221 to 225, further comprising a third manifold or connector having the fluid passage extending in the y-z plane. 227. The flow cell system of any one of claims 221 to 226, wherein the first pump is configured to clean the open landing region by displacing the residual amount of the first reagent away from the open landing region to flow through the clean outlet. 228. A method for preparing a flow cell for a DNA sequencing reaction, the method comprising: (a) providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet comprises an open landing region for receiving one or more reagents, and (ii) one or more channels, the one or more...(a) A channel is disposed between the inlet and the outlet to perform the sequencing reaction; (b) a first reagent of the one or more reagents is openly dispensed into the open landing area so that at least a portion of the first reagent flows from the open landing area into the one or more channels; (c) a gas is introduced into the one or more channels; (d) a second reagent of the one or more reagents is openly dispensed into the open landing area so that at least a portion of the second reagent flows from the open landing area into the one or more channels, thereby removing residual amounts of the first reagent from the one or more channels. 229. A method for preparing a flow cell for a DNA sequencing reaction, the method comprising: (a) providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet includes an open landing region for receiving one or more reagents, and (ii) one or more channels disposed between the inlet and the outlet to perform the sequencing reaction; (b) openly dispensing a first reagent of the one or more reagents into the open landing region such that at least a portion of the first reagent flows from the open landing region into the one or more channels; wherein at least a portion of the open landing region includes a surface coating to facilitate the removal of residual amounts of the first reagent from the open landing region; and (c) openly dispensing a second reagent of the one or more reagents into the open landing region such that at least a portion of the second reagent flows from the open landing region into the one or more channels. 230. A method for sequencing using a flow cell device, the method comprising: (a) providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet includes an open landing region for receiving one or more reagents, and (ii) one or more channels disposed between the inlet and the outlet to perform the sequencing reaction; (b) openly dispensing a first reagent of the one or more reagents into the open landing region such that at least a portion of the first reagent flows from the open landing region to the one or more channels; (c) removing the residual amount of the first reagent from at least a portion of the open landing region by allowing the residual amount of the first reagent to flow through a clean outlet of the flow cell device; and (d) openly dispensing a second reagent of the one or more reagents into the open landing region such that at least a portion of the second reagent flows from the open landing region to the one or more channels. 231. A method for manufacturing a flow cell device, the method comprising: obtaining one or more substrates;The method comprises: generating one or more channels in one or more substrates, wherein the one or more channels are configured to allow fluid or an air gap between the fluid and another fluid to flow through the one or more channels; forming an inlet comprising an aperture in one of the one or more substrates and an open landing region, wherein the inlet is fluidly connected to the one or more channels; forming an outlet, wherein the outlet is fluidly connected to the one or more channels; coating at least a portion of the surface of the one or more channels with a first coating, wherein the surface is configured to be dried and re-wetted during a DNA sequencing run; and fixing one of the substrates together. 232. A method for manufacturing a flow cell apparatus, the method comprising: obtaining one or more substrates; generating one or more channels in the one or more substrates; forming an inlet comprising an aperture in one of the one or more substrates and an open landing region, wherein the inlet is fluidly connected to the one or more channels; coating at least a portion of the surface of the one or more channels with a first coating; covering at least a portion of the open landing region with a second coating; and fixing one of the substrates together. Claims 14 / 22, Page 15, CN 121057626 A 233. A method for manufacturing a flow cell apparatus, the method comprising: obtaining one or more substrates; forming an inlet, the inlet including an aperture and an open landing region in one of the one or more substrates; generating one or more channels in the one or more substrates; forming an outlet in the one or more substrates, wherein the inlet and the outlet are fluidly connected to the one or more channels; forming a clean outlet in the one or more substrates, wherein the clean outlet is fluidly connected to the inlet, and wherein the clean outlet is closer to the inlet than to the outlet; and fixing one of the one or more substrates together. 234. The method of any one of claims 231 to 233, wherein the one or more channels are configured to allow the air gap to flow through the one or more channels between allowing the first reagent and the second reagent to flow through the one or more channels. 235. The method of any one of claims 231 to 234, wherein the one or more channels are configured to allow the air gap to flow through the one or more channels during a DNA sequencing run. 236. The method of any one of claims 231 to 235, wherein the one or more channels are configured to allow air gap flow from the inlet through the one or more channels. 237. The method of any one of claims 231 to 236, wherein the one or more channels are configured toAllowing air gap flow through the one or more channels to facilitate reduced contamination of the second reagent by the first reagent during a DNA sequencing run. 238. The method of any one of claims 231 to 237, wherein the one or more channels are configured to allow air gap flow through the one or more channels to reduce the minimum amount of the first reagent, the second reagent, or washing reagent required for a DNA sequencing run. 239. The method of any one of claims 231 to 238, wherein one of the one or more channels comprises one or more surfaces. 240. The method of claim 239, wherein the one or more surfaces comprises an inner surface. 241. The method of claim 239, wherein the one or more surfaces comprises an outer surface. 242. The method of claim 239, wherein the one or more surfaces comprises an inner top surface, an inner bottom surface, or both. 243. The method of claim 239, wherein the one or more surfaces comprises an outer top surface, an outer bottom surface, or both. 244. The method of claim 239, wherein the one or more surfaces comprises a planar surface. 245. The method of claim 239, wherein the one or more surfaces are passivated. 246. The method of claim 239, wherein the one or more surfaces are passivated with a coating that immobilizes the surface to capture primers, nucleic acid template molecules, or both, to capture polynucleotides. 247. The method of claim 239, wherein the one or more surfaces contain polynucleotides captured thereon. 248. The method of any one of claims 231 to 247, wherein the air gap is configured to dry at least a portion of the one or more surfaces of the one or more channels. 249. The method of any one of claims 239 to 248, wherein the air gap does not impair the chemical function of the one or more surfaces. 250. The method of any one of claims 231 to 249, wherein the coating of the one or more surfaces comprises at least one hydrophilic polymer coating. 251. The method of any one of claims 231 to 250, wherein the coating of the one or more surfaces comprises a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating. 252. The method according to any one of claims 239 to 251, wherein the one or more surfaces comprise at least one discrete region, the at least one discrete region comprising a plurality of cloned amplified sample nucleic acid molecules, the plurality of cloned amplified sample nucleic acid molecules having been annealed to a plurality of attached oligonucleotide molecules.253. The method of claim 251, wherein the water contact angle of the at least one hydrophilic polymer coating does not exceed about 50 degrees. 254. The method of claim 252, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a tandem polymer, the tandem polymer being annealed to at least one of the plurality of attached oligonucleotide molecules. 255. The method of claim 251, wherein the at least one hydrophilic polymer coating comprises PEG. 256. The method of claim 251, wherein the one or more surfaces further comprises a second hydrophilic polymer coating. 257. The method of claim 251, wherein the at least one hydrophilic polymer coating comprises a branched hydrophilic polymer. 258. The method of claim 257, wherein the branched hydrophilic polymer comprises at least eight branches. 259. The method of claim 252, wherein the at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule, the single-stranded multimeric nucleic acid molecule comprising a repeating sequence of regularly occurring monomeric units. 260. The method of claim 259, wherein the single-stranded multimeric nucleic acid molecule has a length of at least 10 kilobases. 261. The method of claim 259, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules further comprises a double-stranded monomer copy of the regularly occurring monomeric unit. 262. The method of claim 251, wherein the plurality of oligonucleotide molecules are present at various locations on the one or more surfaces with a substantially uniform surface density. 263. The method of claim 251, wherein the plurality of oligonucleotide molecules are present at a local surface density of at least 100,000 molecules / μm² in a first region on the one or more surfaces, and at a second local surface density in a second region on the one or more surfaces. 264. The method of claim 246, wherein the coating comprises: (a) a first layer comprising monolayer polymer molecules tethered to one or more surfaces of the substrate; (b) a second layer comprising a second monolayer polymer molecules tethered to the polymer molecules of the first layer; and (c) a third layer comprising a third monolayer polymer molecules tethered to the polymer molecules of the second layer, wherein at least one of the first layer, the second layer, or the third layer comprises branched polymer molecules. Claims 16 / 22 pages 17 CN 121057626 A265. The method of claim 264, wherein the third layer further comprises oligonucleotides tethered to the polymer molecules of the third layer. 266. The method of claim 265, wherein the oligonucleotides tethered to the polymer molecules of the third layer are distributed throughout the third layer at multiple depths. 267. The method of claim 264, wherein the coating further comprises: (a) a fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer, and (b) a fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer. 268. The method of claim 267, wherein the polymer molecules of the fifth layer further comprise oligonucleotides tethered to the polymer molecules of the fifth layer. 269. The method of claim 268, wherein the oligonucleotides tethered to the polymer molecules of the fifth layer are distributed throughout the fifth layer at multiple depths. 270. The method of claim 250, wherein the at least one hydrophilic polymer coating comprises: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran. 271. The method of claim 252, wherein when the cloned amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine dye-3, the ratio of the fluorescence intensity of the cloned amplified, cyanine dye-3 labeled sample nucleic acid molecule or its complementary sequence to the fluorescence intensity of the nonspecific cyanine dye-3 adsorption background (B gap) shown in the images of the one or more surfaces is at least 3:1. 272. The method of claim 271, wherein the ratio of the fluorescence intensity of the cloned, cyanine-3-labeled sample nucleic acid molecule or its complementary sequence shown in the images of the one or more surfaces to the fluorescence intensity of the combination of nonspecific cyanine-3 dye adsorption background and nonspecific amplification background (B-gap + B-endoplasm) is at least 3:1. 273. The method of claim 252, wherein when the cloned, cyanine-3-labeled sample nucleic acid molecule or its complementary sequence is labeled with cyanine-3, the ratio of the fluorescence intensity of the cloned, cyanine-3-labeled sample nucleic acid molecule or its complementary sequence shown in the images of the one or more surfaces to the fluorescence intensity of the nonspecific dye adsorption background (B-gap) is at least 3:1.The intensity ratio is at least 5:1. 274. The method of claim 273, wherein the ratio of the fluorescence intensity of the cloned, amplified, cyanine-3-labeled sample nucleic acid molecule or its complementary sequence exhibited by the image of said one or more surfaces to the fluorescence intensity of the combination of nonspecific cyanine-3 dye adsorption background and nonspecific amplification background (B interstitial + B endoplasm) is at least 5:1. 275. The method of claim 252, wherein, when the cloned, amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine-3, and the fluorescence image of said one or more surfaces is acquired under non-signal saturation conditions using an inverted microscope equipped with a 20× objective lens, NA=0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for cyanine-3 emission, and a camera while said one or more surfaces are immersed in buffer, the contrast-to-noise ratio (CNR) exhibited by said fluorescence images is at least 20. 276. The method of claim 251, wherein said plurality of oligonucleotide molecules are present at a surface density of at least 1,000 molecules / m². Claims 17 / 22 Page 18 CN 121057626 A 277. The method of any one of claims 231 to 276, wherein the first reagent is configured to wet the one or more surfaces of the one or more channels. 278. The method of any one of claims 231 to 277, wherein the second reagent is configured to rewet the one or more surfaces of the one or more channels after drying the one or more surfaces at least partially through the air gap. 279. The method of any one of claims 231 to 278, wherein the flow cell system includes the flow cell device, wherein the flow cell system further includes a fluid control device comprising: (a) a first pump coupled to the outlet; and (b) a dispenser configured to openly dispense one or more reagents to the inlet. 280. The method of any one of claims 231 to 279, wherein the first pump or the second pump is configured to introduce the air gap through the inlet and cause the air gap to flow at least partially through the one or more channels. 281. The method of any one of claims 231 to 280, wherein the air gap comprises air. 282. The method of any one of claims 231 to 280, wherein the air gap comprises dry air. 283. The method of any one of claims 231 to 280, wherein the air gap comprises one or more inert gases. 284. The method of any one of claims 231 to 280, wherein the air gap comprises one or more active gases.285. The method of any one of claims 231 to 284, wherein the first reagent or the second reagent comprises a liquid. 286. The method of any one of claims 231 to 285, wherein the first reagent or the second reagent lacks bubbles larger than a predetermined size. 287. The method of any one of claims 246 to 286, wherein the coating comprises a liquid-repellent coating. 288. The method of any one of claims 246 to 286, wherein the coating comprises a superhydrophobic coating. 289. The method of any one of claims 246 to 286, wherein the coating comprises a smooth liquid-filled porous surface (SLIPS). 290. The method of any one of claims 246 to 286, wherein the coating comprises a smooth superhydrophobic covalently attached liquid (SOCAL) coating. 291. The method of any one of claims 246 to 286, wherein the coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates. 292. The method of any one of claims 246 to 286, wherein the coating is formed by impregnating a lubricant in one or more porous surfaces. 293. The method of claim 292, wherein the lubricant comprises a liquid with a surface energy of less than about 20 mJ / m². 294. The method of claim 292, wherein the lubricant comprises silicone oil. 295. The method of any one of claims 246 to 286, wherein the surface energy of the coating is less than about 20 mJ / m². 296. The method of any one of claims 246 to 286, wherein the coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers. 297. The method of claim 296, wherein the one or more brine monomers comprises dimethyldimethoxysilane. Claims 18 / 22 pages 19 CN 121057626 A 298. The method of any one of claims 231 to 297, wherein the open landing region is fluidly connected to the one or more channels. 299. The method of any one of claims 231 to 297, wherein the open landing region is fluidly connected to one of the one or more channels. 300. The method of any one of claims 231 to 297, wherein the open landing region is located on a bottom substrate of the one or more substrates. 301. The method of any one of claims 231 to 300, wherein the inlet comprises a hole in a top substrate of the one or more substrates. 302. The method of claim 301, wherein the hole in the top substrate is positioned above at least a portion of the open landing area.303. The method of any one of claims 279 to 302, wherein the dispenser is configured to openly dispense the one or more reagents through the orifice to the open landing region. 304. The method of any one of claims 279 to 302, wherein the dispenser is configured to openly dispense the one or more reagents from the tip of the dispenser to the open landing region. 305. The method of any one of claims 279 to 302, wherein the dispenser is configured to openly dispense the one or more reagents from the tip of the dispenser to the open landing region without requiring a conduit between the dispenser and the open landing region. 306. The method of claim 305, wherein at least a portion of the tip of the dispenser contacts the open landing region. 307. The method of claim 305, wherein the tip of the dispenser does not contact the open landing region. 308. The method of any one of claims 231 to 307, wherein the flow cell device further includes a cleaning outlet in one or more substrates. 309. The method of claim 308, wherein the cleaning outlet is fluidly connected to the inlet. 310. The method of claim 308, wherein the cleaning outlet is fluidly connected to the open landing area. 311. The method of claim 308, wherein the cleaning outlet is located in a top or bottom substrate of the one or more substrates. 312. The method of claim 308, wherein the cleaning outlet comprises a side port on the one or more substrates, wherein the side port: (a) extends at least along a direction perpendicular to or substantially perpendicular to the x-direction; (b) extends at least along a direction perpendicular to or substantially perpendicular to the y-direction; (c) extends at least along a direction perpendicular to or substantially perpendicular to the z-direction; (d) extends at least along a direction inclined to the x-direction; (e) extends at least along a direction inclined to the y-direction; or (f) extends at least along a direction inclined to the z-direction. 313. The method of claim 308, wherein the cleaning outlet is configured to be coupled to the first pump or the second pump. 314. The method of any one of claims 231 to 313, wherein the one or more channels comprise microfluidic channels. Claims 19 / 22, page 20, CN 121057626 A 315. The method according to any one of claims 231 to 314, wherein the one or more surfaces are coated with fluorescent beads, the fluorescent beads being chemically fixed to the one or more surfaces. 316. The method according to claim 315, wherein the fluorescent beads are covalently attached to the one or more surfaces.317. The method of any one of claims 231 to 316, wherein the gap between the inner top surface and the inner bottom surface is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. 318. The method of any one of claims 231 to 316, wherein the height of the one or more channels is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. 319. The method of any one of claims 247 to 318, wherein the polynucleotides captured thereon are configured for imaging in a sequencing cycle. 320. The method of any one of claims 231 to 319, wherein the one or more substrates comprise a top substrate and a bottom substrate. 321. The method of claim 320, wherein the one or more channels are defined between the top substrate and the bottom substrate. 322. The method of claim 320, wherein the one or more channels are at least partially defined in the top surface of the bottom substrate. 323. The method of claim 320, wherein the one or more channels are at least partially defined in the bottom surface of the top substrate. 324. The method of claim 320, wherein the one or more substrates further comprise an intermediate substrate. 325. The method of claim 324, wherein the one or more channels are at least partially defined in the intermediate substrate. 326. The method of any one of claims 231 to 325, wherein the one or more substrates comprise glass or plastic. 327. The method of any one of claims 231 to 326, wherein at least a portion of the carrier is transparent. 328. The method of any one of claims 231 to 327, wherein at least a portion of the one or more substrates is transparent. 329. The method of any one of claims 231 to 328, wherein the carrier is solid-phase. 330. The method of any one of claims 231 to 329, wherein the one or more channels comprise 1, 2, 3, 4, 5, 6, 7, or 8 channels. 331. The method of any one of claims 231 to 330, wherein the one or more channels comprise 2, 4, 6, 8, or 10 channels. 332. The method of any one of claims 231 to 331, wherein the lane length of each of the one or more channels is less than about 70 mm, 75 mm, 80 mm, or 90 mm.333. The method of any one of claims 231 to 331, wherein the lane width of each of the one or more channels is less than about 10 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, or 3 mm. 334. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating, the second coating comprising a smooth coating. 335. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating, the second coating comprising a liquid-repellent coating. 336. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating, the second coating comprising a superhydrophobic coating. 337. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating, the second coating comprising a smooth liquid-filled porous surface (SLIPS). 338. The method of any one of claims 231 to 333, wherein at least a portion of the open landing region comprises a second coating comprising a smooth, superhydrophobic, covalently attached liquid (SOCAL) coating. 339. The method of any one of claims 231 to 333, wherein at least a portion of the open landing region comprises a second coating comprising a liquid-like polymer brush surface covalently attached to the one or more substrates. 340. The method of any one of claims 231 to 333, wherein at least a portion of the open landing region comprises a second coating comprising impregnating a lubricant in a porous surface to produce a coating with a surface energy below about 20 mJ / m². 341. The method of any one of claims 231 to 333, wherein at least a portion of the open landing region comprises a second coating comprising an impregnated acid-catalyzed graft polycondensation of one or more brine monomers. 342. The method of claim 341, wherein the one or more brine monomers comprise dimethyldimethoxysilane. 343. The method of any one of claims 231 to 342, wherein the process of using the flow cell apparatus includes removing at least a portion of the first reagent from at least a portion of the one or more channels during a DNA sequencing run. 344. The method of claim 343, wherein the at least a portion of the first reagent remains in the one or more channels during the DNA sequencing run. 345. The method of any one of claims 231 to 344, wherein the first reagent and the second reagent are different.346. The method of any one of claims 231 to 345, wherein at least a portion of the one or more channels comprises more than 40% of the corresponding volume or length of each of the one or more channels. 347. The method of any one of claims 231 to 345, wherein at least a portion of the one or more channels comprises more than half of the corresponding volume or length of each of the one or more channels. 348. The method of any one of claims 231 to 345, wherein at least a portion of the one or more channels comprises more than 60% of the corresponding volume or length of each of the one or more channels. 349. The method of any one of claims 231 to 345, wherein at least a portion of the one or more channels comprises more than 70% of the corresponding volume or length of each of the one or more channels. 350. The method of any one of claims 231 to 345, wherein at least a portion of the one or more channels comprises more than 80% of the corresponding volume or length of each of the one or more channels. 351. The method of any one of claims 231 to 350, wherein the process of using the flow cell device comprises expelling a residual amount of the first reagent or the second reagent from the open landing area through a clean outlet of the flow cell device. 352. The method of claim 351, wherein the clean outlet is configured to allow a residual amount of the first reagent on the open landing area to flow through the clean outlet. Claims 21 / 22 pages 22 CN 121057626 A 353. The method of any one of claims 231 to 352, wherein the flow cell system comprises the flow cell device, wherein the flow cell system further comprises a fluid control device comprising: (a) a first pump fluidly connected to the clean outlet, wherein the first pump or a second pump is fluidly connected to the outlet; and (b) a dispenser configured to openly dispense the one or more reagents to the inlet. 354. The method of claim 353, wherein the first pump is configured to clean the open landing area by expelling a residual amount of the first reagent from the open landing area to flow through the clean outlet. 355. The method of claim 343, wherein the process further comprises: removing at least a portion of the first reagent from at least a portion of the one or more channels by driving fluid from the inlet through the air gap and through at least a portion of the one or more channels. 356. The method of any one of claims 351 to 355, wherein the residual amount of the first reagent on the open landing area comprises a meniscus of the first reagent. ClaimsPage 22 / 22 23 CN 121057626 A Cross-Reference to Applications Related to Flow Cell Apparatus and Its Uses

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 479,158, filed January 9, 2023, and U.S. Provisional Application No. 63 / 502,896, filed May 17, 2023, each of which is incorporated herein by reference in its entirety.

[0002] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication, patent, or patent application is expressly and individually indicated to be incorporated herein by reference in its entirety. In the event of any conflict between terms used herein and terms in the incorporated references, the terms used herein shall prevail. Background Art

[0003] Flow cell apparatuses are used in chemical and biotechnology applications. In next-generation sequencing (NGS) systems, flow cell devices are used to immobilize template nucleic acid molecules derived from biological samples, and then repeated streams of sequencing reagents are introduced to attach labeled nucleotides to specific locations in the template sequence. A series of label signals are detected and decoded to reveal the nucleotide sequence of the template molecule, such as an immobilized or amplified nucleic acid template molecule or a combination thereof attached to the surface of the flow cell.

[0004] Existing NGS flow cells are multilayer structures made of planar surface substrates and other flow cell components, which are then combined to form fluid flow channels. Such flow cells may require expensive, multi-step precision manufacturing techniques to achieve the desired design specifications. On the other hand, inexpensive and readily available single-channel capillaries come in various sizes and shapes, but are generally not suitable for easy handling or compatibility with the repetitive switching of reagents required for applications such as NGS.

[0005] In one aspect, this invention discloses a flow cell apparatus comprising a carrier including one or more substrates, wherein the one or more substrates include an inlet and an outlet, wherein the inlet includes an open landing region; and one or more channels defined by the one or more substrates, wherein the one or more channels are fluidly connected to the inlet and the outlet, wherein the one or more channels are configured to allow fluid or an air gap between the fluid and another fluid to flow through the one or more channels. In some embodiments, the open landing region is at least partially covered with a surface coating. In some embodiments, the one or more channels extend from the inlet to the outlet. In some embodiments, the one or more channels extend along a first direction and between the inlet and the outlet. In some embodiments, the one or more channels are configured to allow the air gap to flow through the one or more channels, wherein the fluid includes a first reagent and the other fluid includes a second reagent.Reagents. In some embodiments, the one or more channels are configured to allow air gaps to flow through the one or more channels during a DNA sequencing run. In some embodiments, the one or more channels are configured to allow air gaps to flow through the one or more channels from the inlet. In some embodiments, the one or more channels are configured to allow air gaps to flow through the one or more channels to facilitate reduced contamination of the first reagent to the second reagent during the DNA sequencing run. In some embodiments, the one or more channels are configured to allow air gaps to flow through the one or more channels to reduce the minimum amount of the first reagent, the second reagent, or the washing reagent used in the DNA sequencing run. In some embodiments, the one or more channels include one or more surfaces. In some embodiments, the one or more surfaces include an inner surface. In some embodiments, the one or more surfaces include an inner top surface, an inner bottom surface, or both. In some embodiments, the one or more surfaces include an outer top surface, an outer bottom surface, or both. In some embodiments, the one or more surfaces include planar surfaces. In some embodiments, the one or more surfaces are passivated. In some embodiments, the one or more surfaces are passivated with a coating that immobilizes the surface to capture primers, nucleic acid template molecules, or both, to capture polynucleotides. In some embodiments, the one or more surfaces contain the polynucleotides coupled thereto. In some embodiments, the air gap is configured to remove moisture or liquid from at least a portion of the one or more surfaces of the one or more channels. In some embodiments, the air gap does not impair the chemical function of the one or more surfaces. In some embodiments, the coating of the one or more surfaces comprises at least one hydrophilic polymer coating. In some embodiments, the coating of the one or more surfaces comprises a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating. In some embodiments, the one or more surfaces comprise at least one discrete region containing a plurality of cloned amplified sample nucleic acid molecules that have been annealed to a plurality of attached oligonucleotide molecules. In some embodiments, the water contact angle of the at least one hydrophilic polymer coating does not exceed about 50 degrees. In some embodiments, at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a tandem mass that has been annealed to at least one of the plurality of attached oligonucleotide molecules. In some embodimentsIn some embodiments, the at least one hydrophilic polymer coating comprises polyethylene glycol (PEG). In some embodiments, the one or more surfaces further comprise a second hydrophilic polymer coating. In some embodiments, the at least one hydrophilic polymer coating comprises a branched hydrophilic polymer. In some embodiments, the branched hydrophilic polymer comprises at least eight branches. In some embodiments, the at least one cloned amplified sample nucleic acid molecule in the plurality of cloned amplified sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule, the single-stranded multimeric nucleic acid molecule comprising a repeating sequence of regularly occurring monomeric units. In some embodiments, the length of the single-stranded multimeric nucleic acid molecule is at least 10 kilobases. In some embodiments, the at least one cloned amplified sample nucleic acid molecule in the plurality of cloned amplified sample nucleic acid molecules further comprises a double-stranded monomeric copy of the regularly occurring monomeric units. In some embodiments, the plurality of oligonucleotide molecules are present at various locations on the one or more surfaces with a substantially uniform surface density. In some embodiments, the plurality of oligonucleotide molecules are present at a first location on the one or more surfaces with a local surface density of at least 100,000 molecules / μm², and at a second location on the one or more surfaces with a second local surface density. In some embodiments, the coating comprises: a first layer comprising a monolayer of polymer molecules tethered to a surface of a substrate in one or more substrates; a second layer comprising a second monolayer of polymer molecules tethered to the polymer molecules of the first layer; and a third layer comprising a third monolayer of polymer molecules tethered to the polymer molecules of the second layer, wherein at least one of the first, second, or third layers comprises branched polymer molecules. In some embodiments, the third layer further comprises oligonucleotides tethered to the polymer molecules of the third layer. In some embodiments, the oligonucleotides tethered to the polymer molecules of the third layer are distributed at multiple depths throughout the third layer. In some embodiments, the coating further comprises: a fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer; and a fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer. In some embodiments, the polymer molecules of the fifth layer further comprise oligonucleotides tethered to the polymer molecules of the fifth layer. In some embodiments, the oligonucleotides tethered to the polymer molecules of the fifth layer are described in the specification, page 2 / 105, 25 CN 121057626 A.Multiple depths are distributed throughout the fifth layer. In some embodiments, the at least one hydrophilic polymer coating comprises: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran.

[0006] In some embodiments, when the sample nucleic acid molecules or their complementary sequences of the plurality of cloned amplifications are labeled with cyanine dye-3, the ratio of the fluorescence intensity of the sample nucleic acid molecules or their complementary sequences labeled with cyanine dye-3 to the fluorescence intensity of the nonspecific cyanine dye-3 adsorption background (B gap) shown in the images of the one or more surfaces is at least 3:1. In some embodiments, the ratio of fluorescence intensity of the amplified sample nucleic acid molecules or their complementary sequences labeled with cyanine dye-3 to the fluorescence intensity of the combination of non-specific cyanine dye-3 adsorption background and non-specific amplification background (Bg gap + Bg endoplasm) shown in the images of the one or more surfaces is at least 3:1. In some embodiments, when the amplified sample nucleic acid molecules or their complementary sequences are labeled with cyanine dye-3, the ratio of fluorescence intensity of the amplified sample nucleic acid molecules or their complementary sequences labeled with cyanine dye-3 to the fluorescence intensity of the non-specific dye adsorption background (Bg gap) shown in the images of the one or more surfaces is at least 5:1. In some embodiments, the ratio of fluorescence intensity of the amplified sample nucleic acid molecules or their complementary sequences labeled with cyanine dye-3 to the fluorescence intensity of the combination of non-specific cyanine dye-3 adsorption background and non-specific amplification background (Bg gap + Bg endoplasm) shown in the images of the one or more surfaces is at least 5:1. In some embodiments, when the plurality of cloned and amplified sample nucleic acid molecules or their complementary sequences are labeled with cyanine dye-3, and when the fluorescence images of the one or more surfaces are acquired under non-signal saturation conditions using an inverted microscope equipped with a 20× objective lens, NA = 0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for cyanine dye-3 emission, and a camera, the fluorescence images exhibit a contrast-to-noise ratio (CNR) of at least 20. In some embodiments, the plurality of oligonucleotide molecules are arranged at a surface density of at least 1,000 molecules / m².The presence of a certain degree of moisture content is present. In some embodiments, the first reagent is configured to wet the one or more surfaces of the one or more channels. In some embodiments, the second reagent is configured to rewet the one or more surfaces of the one or more channels after at least a portion of the moisture or liquid has been removed from the one or more surfaces of the one or more channels. In some embodiments, the air gap contains air. In some embodiments, the air gap contains dry air. In some embodiments, the air gap contains one or more inert gases. In some embodiments, the air gap contains one or more active gases. In some embodiments, the first reagent or the second reagent contains a liquid. In some embodiments, the first reagent or the second reagent does not contain bubbles larger than a predetermined size. In some embodiments, the coating comprises a liquid-repellent coating. In some embodiments, the coating comprises a superhydrophobic coating. In some embodiments, the coating comprises a smooth liquid-injected porous surface (SLIPS). In some embodiments, the coating comprises a smooth superhydrophobic covalently attached liquid (SOCAL) coating. In some embodiments, the coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates. In some embodiments, the coating is formed by impregnating a lubricant in one or more porous surfaces. In some embodiments, the lubricant comprises a liquid with a surface energy of less than about 20 mJ / m². In some embodiments, the lubricant comprises a silicone oil. In some embodiments, the surface energy of the coating is less than about 20 mJ / m². In some embodiments, the coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers. In some embodiments, the one or more brine monomers comprise dimethyldimethoxysilane. In some embodiments, the open landing region is fluidly connected to one or more channels. In some embodiments, the open landing region is fluidly connected to one of the one or more channels. In some embodiments, the open landing region is fluidly connected to two or more of the one or more channels. In some embodiments, the open landing region is located on the bottom substrate of one or more substrates. In some embodiments, the inlet comprises a hole in the top substrate of one or more substrates. In some embodiments, the hole in the top substrate is positioned at least partially above the open landing region. In some embodiments, the flow cell device is configured to allow a dispenser to openly dispense one or more reagents through the hole to the open landing region. In some embodiments, the dispenser is configured to openly dispense the one or more reagents from the tip of the dispenser.Dispensing to the open landing area. In some embodiments, the dispenser is configured to dispense the one or more reagents openly from the tip of the dispenser to the open landing area without the need for conduit between the dispenser and the open landing area. In some embodiments, at least a portion of the tip of the dispenser contacts the open landing area. In some embodiments, the tip of the dispenser does not contact the open landing area. In some embodiments, the flow cell device further includes a cleaning outlet in one or more substrates. In some embodiments, the cleaning outlet is fluidly connected to the inlet. In some embodiments, the cleaning outlet is fluidly connected to the open landing area. In some embodiments, the cleaning outlet is located below the open landing area. In some embodiments, the cleaning outlet is located in a top or bottom substrate of one or more substrates. In some embodiments, the cleaning outlet includes a side port on one or more substrates, wherein the side port: extends at least along a direction perpendicular to or substantially perpendicular to the x-direction; extends at least along a direction perpendicular to or substantially perpendicular to the y-direction; extends at least along a direction perpendicular to or substantially perpendicular to the z-direction; extends at least along a direction inclined to the x-direction; extends at least along a direction inclined to the y-direction; or extends at least along a direction inclined to the z-direction. In some embodiments, the cleaning outlet is configured to be coupled to a first pump or a second pump. In some embodiments, the one or more channels comprise one or more microfluidic channels. In some embodiments, the one or more surfaces are coated with fluorescent beads, the fluorescent beads being chemically immobilized to the one or more surfaces. In some embodiments, the fluorescent beads are covalently attached to the one or more surfaces. In some embodiments, the gap between the inner top surface and the inner bottom surface is approximately 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. In some embodiments, the height of the one or more channels is approximately 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. In some embodiments, the polynucleotides captured thereon are configured for imaging during a sequencing cycle. In some embodiments, the one or more substrates comprise a top substrate and a bottom substrate. In some embodiments, the one or more channels are defined between the top substrate and the bottom substrate. In some embodiments, the one or more channels are at least partially defined in the top surface of the bottom substrate. In some embodiments, the one or more channels are at least partially defined in the bottom surface of the top substrate. In some embodiments, the one or more channels are...The plurality of substrates further includes an intermediate substrate. In some embodiments, the one or more channels are at least partially defined in the intermediate substrate. In some embodiments, the one or more substrates comprise glass or plastic. In some embodiments, at least a portion of the carrier is transparent. In some embodiments, at least a portion of the one or more substrates is transparent. In some embodiments, the carrier is solid-phase. In some embodiments, the one or more channels comprise 1, 2, 3, 4, 5, 6, 7, or 8 channels. In some embodiments, the one or more channels comprise 2, 4, 6, 8, or 10 channels. In some embodiments, the lane length of each of the one or more channels is less than about 70 mm, 75 mm, 80 mm, or 90 mm. In some embodiments, the lane width of each of the one or more channels is less than about 10 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, or 3 mm. In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising a smooth coating. In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising a liquid repellent coating. In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising a superhydrophobic coating. In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising a smooth liquid-filled porous surface (SLIPS). In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising a smooth superhydrophobic covalently attached liquid (SOCAL) coating. In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising a liquid-like polymer brush surface covalently attached to the one or more substrates. In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising impregnating a lubricant in a porous surface to produce a second surface coating with a surface energy below about 20 mJ / m². In some embodiments, at least a portion of the open landing area is covered with a second surface coating comprising an acid-catalyzed graft polycondensation of one or more brine monomers. In some embodiments, the one or more brine monomers comprise dimethyldimethoxysilane. In some embodiments, the flow cell device is configured to allow cleaning of at least a portion of the first reagent from at least a portion of the one or more channels during a DNA sequencing run.In some embodiments, the flow cell device is configured to allow at least a portion of the first reagent to remain in the one or more channels. In some embodiments, the first reagent and the second reagent are different. In some embodiments, at least a portion of the one or more channels occupies more than 40% of the corresponding volume or length of each of the one or more channels. In some embodiments, at least a portion of the one or more channels occupies more than half of the corresponding volume or length of each of the one or more channels. In some embodiments, at least a portion of the one or more channels occupies more than 60% of the corresponding volume or length of each of the one or more channels. In some embodiments, at least a portion of the one or more channels occupies more than 70% of the corresponding volume or length of each of the one or more channels. In some embodiments, at least a portion of the one or more channels occupies more than 80% of the corresponding volume or length of each of the one or more channels. In some embodiments, the clean outlet is configured to allow residual amounts of the first reagent on the open landing area to flow through the clean outlet. In some embodiments, the residual amount of the first reagent on the open landing area comprises a meniscus of the first reagent. In some embodiments, the flow cell device further includes one or more seals positioned on the one or more substrates. In some embodiments, a first portion of one or more channels includes a first z-position, and a second portion of the channel includes a second z-position different from the first z-position. In some embodiments, the first portion of the channel includes one or more first imaging surfaces. In some embodiments, the second portion of the channel includes one or more second imaging surfaces. In some embodiments, the top substrate or the bottom substrate includes one or more substrate layers. In some embodiments, the top substrate includes a first thickness above the first portion of the channel and a second thickness around the second portion of the channel. In some embodiments, the second thickness is greater than the first thickness. In some embodiments, the second thickness is 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the first thickness. In some embodiments, the bottom substrate includes a third thickness above the first portion of the channel and a fourth thickness above the second portion of the channel. In some embodiments, the fourth thickness is greater than the third thickness. In some embodiments, the fourth thickness is 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the third thickness. In some embodiments, the one or more seals include aOne or more mechanical seals. In some embodiments, the one or more seals comprise one or more gaskets. In some embodiments, the cleaning outlet is configured to remove fluid from the one or more channels. In some embodiments, the cleaning outlet is in sealed fluid connection to a pump or vacuum device. In some embodiments, the cleaning outlet is configured to direct fluid or gas to the one or more channels. In some embodiments, the bottom substrate comprises glass, plastic, or both. In some embodiments, the one or more seals comprise a first seal having a thickness along the z-direction equivalent to the thickness of the top substrate in the second portion. In some embodiments, the one or more seals comprise a second seal having a thickness along the z-direction equivalent to the thickness of the bottom substrate in the second portion. In some embodiments, the thickness of the second seal along the z-direction is greater than the thickness of the bottom substrate in the first portion.

[0007] In some embodiments, the flow cell device further comprises a frame covering at least a portion of the one or more substrates. In some embodiments, the frame is mechanically secured to the one or more seals. In some embodiments, the frame comprises plastic. In some embodiments, the one or more seals interface with a manifold or connector to allow sealed fluid communication between the manifold or connector and the one or more channels. In some embodiments, the manifold or connector includes one or more fluid passages. In some embodiments, the one or more fluid passages are in fluid communication with the one or more channels. In some embodiments, the one or more fluid passages are in fluid communication with the open landing area. In some embodiments, the manifold or connector is configured to seal fluid communication with the one or more channels by applying a pressure thereon that meets a predetermined threshold. In some embodiments, the one or more fluid passages extend along a y-axis, and wherein the pressure is applied along the y-axis. In some embodiments, the one or more fluid passages extend along an x-axis, and wherein the pressure is applied along the x-axis. In some embodiments, the manifold or connector includes a coupling interface that directly contacts an end of the flow pool device. In some embodiments, the manifold or connector includes a coupling interface that contacts an end of the flow pool device with the one or more seals therebetween. In some embodiments, the manifold or connector includes a coupling interface that contacts an end of the flow pool device with an adhesive therebetween. In some embodiments, the manifold or connector includes an open area located within the one or more flow pools.At the end of a fluid passage in the body pathway. In some embodiments, the open region is fluidly connected to the open landing region. In some embodiments, the flow cell device further includes one or more reference features configured to position the flow cell device relative to the manifold or the connector, sample stage, or sequencing system. In some embodiments, the one or more reference features include at least one alignment feature positioned at a center point along the x-axis. In some embodiments, the one or more reference features include at least one alignment feature positioned at or near the end of one or more substrates along the y-axis. In some embodiments, the one or more reference features include a cavity extending through the one or more substrates and configured to engage with a pin. In some embodiments, the one or more reference features include a groove extending through the one or more substrates and configured to engage with a pin. In some embodiments, the manifold or the connector includes a top portion or a bottom portion extending beyond the one or more substrates along the z-axis and covering at least a portion of the one or more substrates in the x-y plane. In some embodiments, the top portion or the bottom portion is located at the first portion, the second portion, or both of the one or more channels. In some embodiments, the top portion or the bottom portion includes one or more alignment features configured to align the top portion or the bottom portion with the flow cell device. In some embodiments, the top portion or the bottom portion includes one or more alignment features configured to align the top portion or the bottom portion with the flow cell device along the z-axis or along the y-axis. In some embodiments, the flow cell device further includes one or more tubes that interface with the manifold or the connector and the flow cell device. In some embodiments, each of the one or more tubes includes a wall surrounding a lumen. In some embodiments, the lumen is in fluid communication with one or more channels of the flow cell device and one or more fluid passages of the manifold or the connector. In some embodiments, at least a portion of the one or more tubes is embedded in the one or more substrates. In some embodiments, each of the one or more tubes is coupled to the manifold or the connector, thereby enabling fluid communication therebetween. In some embodiments, the one or more seals include a protective sleeve seal, the protective sleeve seal being in...The seal covers at least a portion of the flow cell device in the x-y plane and one end of the flow cell device in the x-z plane. In some embodiments, the one or more seals comprise a flexible material that deforms under pressure satisfying a predetermined threshold. In some embodiments, the one or more seals comprise an L-shaped seal extending along the z-axis and the y-axis. In some embodiments, the L-shaped seal extends along the y-axis and into a corresponding channel in one or more channels. In some embodiments, pressure or force is applied to the L-shaped seal along the y-axis to seal fluid communication between the flow cell device and the manifold. In some embodiments, the one or more seals are configured to interface with the manifold or connector, thereby allowing sealed fluid communication between the flow cell device and the manifold. In some embodiments, the one or more seals comprise a membrane seal covering at least a portion of the flow cell device and at least a portion of the manifold or connector, thereby sealing fluid communication therebetween. In some embodiments, the membrane seal comprises a flat washer placed on top of the top surface of the top substrate, a flat washer placed below the bottom surface of the bottom substrate, or both. In some embodiments, the membrane seal extends in the x-y plane. In some embodiments, the manifold or connector includes a finger-cut region located between two channels of the flow cell apparatus. In some embodiments, the manifold or connector includes a seal placed in the finger-cut region and configured to seal fluid communication between the two channels. In some embodiments, the manifold or connector includes a fluid passage having an outlet exiting the manifold in a plane orthogonal to the y-axis, x-axis, or z-axis. In some embodiments, the top substrate or the bottom substrate includes one or more inclined ends. In some embodiments, the tip of one of the inclined ends presses against the one or more seals. In some embodiments, each of the one or more inclined ends interfaces with an inclined manifold or connector. In some embodiments, the one or more inclined ends include a first acute-angled ramp relative to the y-axis. In some embodiments, the inclined manifold or connector includes a second acute-angled ramp relative to the y-axis. In some embodiments, the first acute-angled ramp is different from the second acute-angled ramp. In some embodiments, the first acute-angled ramp is the same as the second acute-angled ramp. In some embodiments, the inclined manifold or connector includes a ramp complementary to the inclined end of the flow pool device. In some embodiments, theOne or more seals include diagonal washers having fluid passages extending in the y-z plane. In some embodiments, the diagonal washers, the manifold, or the connector interfaces with an end of the top substrate and a top surface of the bottom substrate. In some embodiments, the diagonal washers, the manifold, or the connector interfaces with an end of the bottom substrate and a top inner surface of the top substrate. In some embodiments, the diagonal washer manifold or the connector allows sealed fluid communication from the fluid passages to the one or more channels when the force or pressure includes a y-axis component satisfying a first threshold and a z-axis component satisfying a second threshold. In some embodiments, the top substrate and the bottom substrate are offset from each other at least laterally along the y-axis. In some embodiments, at least a portion of the manifold or the connector is fixedly attached to the bottom inner surface of the bottom substrate. In some embodiments, the flow cell device further includes an intermediary layer configured to define the one or more channels between the top substrate and the bottom substrate. In some embodiments, the top substrate and the bottom substrate are not directly fixedly attached to each other. In some embodiments, at least a portion of the manifold or the connector is fixedly attached to the top inner surface of the top substrate. In some embodiments, as described on page 7 / 105 of CN 121057626 A, the diagonal washer, the fluid passage of the manifold or the connector extends at least along the y-axis. In some embodiments, the manifold or the connector further includes an open well leading to a second open landing region, wherein the second open landing region is configured to receive reagent from a dispensing tip. In some embodiments, the open well of the manifold or the connector is in fluid communication with the one or more channels. In some embodiments, the second open landing region of the manifold or the connector is in fluid communication with the inlet of the one or more channels. In some embodiments, the one or more seals include a thermoplastic connector and a thermoplastic seal mounted on the thermoplastic connector. In some embodiments, the thermoplastic seal is deformable under pressure changes, temperature changes, or both. In some embodiments, the thermoplastic seal comprises one or more materials different from one or more materials of the thermoplastic connector. In some embodiments, the one or more seals include a first connector having a top portion that is slidable on a top surface of the top substrate. In some embodiments, the one or more seals include a second connector having a bottom portion that is slidable on a bottom surface of the bottom substrate.The top portion is connected to a first side portion of the first connector, the first side portion being configured to interface with an end of the flow pool device in the x-z plane. In some embodiments, the bottom portion is connected to a second side portion of the second connector, the second side portion being configured to interface with an end of the flow pool device in the x-z plane. In some embodiments, pressure or force satisfying a predetermined threshold on the first and second side portions is configured to cause the first and second connectors to slide relative to the flow pool device under deformation, thereby enabling the one or more channels to be in sealing communication with a fluid passage defined between the top and bottom connectors. In some embodiments, the inlet includes a port that opens at the bottom surface of the bottom substrate. In some embodiments, the port is in fluid communication with the one or more channels of the connector and the fluid passage. In some embodiments, the one or more seals comprise a semi-rigid or deformable material that deforms under pressure or force. In some embodiments, the semi-rigid or deformable material is configured to return to its shape before deformation when the pressure or force is removed. In some embodiments, the one or more seals comprise a gasket, a second connector, a second manifold, or a component thereof, or a combination thereof. In some embodiments, the flow cell device further includes a force application mechanism controlled by computer-readable instructions executable on a computer processor. In some embodiments, the second manifold, the second connector, or the one or more seals are connected to the force application mechanism, thereby allowing connection to or disconnection from the flow cell device.

[0008] On the other hand, a flow cell system is disclosed herein, comprising: the flow cell device disclosed herein; and a fluid control device. In some embodiments, the fluid control device includes a first pump, a second pump, or both. In some embodiments, the fluid control device includes: a third pump connected to the outlet of the flow cell device; and a dispenser configured to openly dispense the one or more reagents to the inlet of the flow cell device. In some embodiments, the fluid control device includes: a fourth pump fluidly connected to the clean outlet of the flow cell device; a fifth pump, wherein the fourth pump or the fifth pump is fluidly connected to the outlet of the flow cell device; and a dispenser configured to openly dispense the one or more reagents to the inlet of the flow cell device. In some embodiments, the first pump or the second pump is configured to introduce the air gap through the inlet and cause the air gap to flow at least partially through the one or more channels.In some embodiments, the flow cell system further includes a third manifold or connector having the fluid passage extending in the y-z plane. In some embodiments, the first pump is configured to clean the open landing area by displacing the residual amount of the first reagent described on page 31 of specification 8 / 105 (CN 121057626 A) from the open landing area to flow through the clean outlet.

[0009] On the other hand, this document discloses a method for preparing a flow cell for a DNA sequencing reaction, the method comprising: (a) providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet comprises an open landing region for receiving one or more reagents, and (ii) one or more channels disposed between the inlet and the outlet to perform the sequencing reaction; (b) openly dispensing a first reagent of the one or more reagents into the open landing region such that at least a portion of the first reagent flows from the open landing region into the one or more channels; (c) introducing a gas into the one or more channels; and (d) openly dispensing a second reagent of the one or more reagents into the open landing region such that at least a portion of the second reagent flows from the open landing region into the one or more channels, thereby removing a residual amount of the first reagent from the one or more channels.

[0010] On the other hand, this document discloses a method for preparing a flow cell for a DNA sequencing reaction, the method comprising: (a) providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet comprises an open landing region for receiving one or more reagents, and (ii) one or more channels disposed between the inlet and the outlet to perform the sequencing reaction; (b) openly dispensing a first reagent of the one or more reagents into the open landing region such that at least a portion of the first reagent flows from the open landing region into the one or more channels; wherein at least a portion of the open landing region comprises a surface coating to facilitate the removal of residual amounts of the first reagent from the open landing region; and (c) openly dispensing a second reagent of the one or more reagents into the open landing region such that at least a portion of the second reagent flows from the open landing region into the one or more channels.

[0011] On the other hand, this document discloses a method for sequencing using a flow cell apparatus, the method comprising: (a) providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet comprises an open landing region for receiving one or more reagents, and (ii) one or more channels, the one or more channels...(a) dispensing a first reagent of the one or more reagents into the open landing area such that at least a portion of the first reagent flows from the open landing area into the one or more channels; (b) removing the residual amount of the first reagent from the open landing area by allowing the residual amount of the first reagent to flow through the clean outlet of the flow cell device; and (d) dispensing a second reagent of the one or more reagents into the open landing area such that at least a portion of the second reagent flows from the open landing area into the one or more channels.

[0012] On the other hand, this document discloses a method for manufacturing a flow cell device, the method comprising: obtaining one or more substrates; forming one or more channels in the one or more substrates, wherein the one or more channels are configured to allow fluid or an air gap between the fluid and another fluid to flow through the one or more channels; forming an inlet comprising a hole and an open landing area in one of the one or more substrates, wherein the inlet is fluidly connected to the one or more channels; forming an outlet, wherein the outlet is fluidly connected to the one or more channels; coating at least a portion of the surface of the one or more channels with a first coating, wherein the surface is configured to be dried and re-wetted during a DNA sequencing run; and fixing one of the one or more substrates together.

[0013] On the other hand, this document discloses a method for manufacturing a flow cell device, the method comprising: obtaining one or more substrates; forming one or more channels in the one or more substrates; forming an inlet, the inlet comprising an aperture and an open landing area in one of the one or more substrates as described in the specification 9 / 105 page 32 CN 121057626 A, wherein the inlet is fluidly connected to the one or more channels; coating at least a portion of the surface of the one or more channels with a first coating; covering at least a portion of the open landing area with a second coating; and fixing one or more substrates together.

[0014] On the other hand, this document discloses a method for manufacturing a flow cell device, the method comprising: obtaining one or more substrates; forming an inlet, the inlet comprising an aperture and an open landing area in one of the one or more substrates; forming one or more channels in the one or more substrates; forming an outlet in the one or more substrates, wherein the inlet and the outlet are fluidly connected to the one or more channels; forming a cleaning outlet in the one or more substrates, wherein the cleaning outlet is fluidly connected to the inlet, and wherein the cleaning outlet is closer to the inlet than to the inlet.The outlet is closer to the inlet; and one of the substrates of the one or more substrates is fixedly coupled together.

[0015] In some embodiments, the one or more channels are configured to allow the air gap to flow through the one or more channels between allowing the first reagent and the second reagent to flow through the one or more channels. In some embodiments, the one or more channels are configured to allow the air gap to flow through the one or more channels during a DNA sequencing run. In some embodiments, the one or more channels are configured to allow the air gap to flow through the one or more channels from the inlet. In some embodiments, the one or more channels are configured to allow the air gap to flow through the one or more channels to facilitate a reduction in contamination of the first reagent to the second reagent during a DNA sequencing run. In some embodiments, the one or more channels are configured to allow the air gap to flow through the one or more channels to reduce the minimum amount of the first reagent, the second reagent, or the washing reagent required for a DNA sequencing run. In some embodiments, one of the one or more channels includes one or more surfaces. In some embodiments, the one or more surfaces includes an inner surface. In some embodiments, the one or more surfaces includes an outer surface. In some embodiments, the one or more surfaces includes an inner top surface, an inner bottom surface, or both. In some embodiments, the one or more surfaces includes an outer top surface, an outer bottom surface, or both. In some embodiments, the one or more surfaces includes a planar surface. In some embodiments, the one or more surfaces are passivated. In some embodiments, the one or more surfaces are passivated with a coating that immobilizes the surface to capture primers, nucleic acid template molecules, or both, to capture polynucleotides. In some embodiments, the one or more surfaces contain polynucleotides captured thereon. In some embodiments, the air gap is configured to dry at least a portion of the one or more surfaces of the one or more channels. In some embodiments, the air gap does not impair the chemical function of the one or more surfaces. In some embodiments, the coating of the one or more surfaces comprises at least one hydrophilic polymer coating. In some embodiments, the coating of the one or more surfaces comprises a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating. In some embodiments, the one or more surfaces comprise at least one discrete region containing a plurality of cloned amplified sample nucleic acid molecules that have been annealed to a plurality of attached oligonucleotide molecules. In some embodiments, the water contact angle of the at least one hydrophilic polymer coating does not exceed about 50 degrees. In some embodiments, the plurality of cloned amplified sample nucleic acids...At least one clone-amplified sample nucleic acid molecule in the plurality of clonal amplified samples contains a tandem polymer, said tandem polymer annealed to at least one of the plurality of attached oligonucleotide molecules. In some embodiments, the at least one hydrophilic polymer coating contains PEG. In some embodiments, the one or more surfaces further contain a second hydrophilic polymer coating. In some embodiments, the at least one hydrophilic polymer coating contains a branched hydrophilic polymer. In some embodiments, the branched hydrophilic polymer contains at least 8 branches. In some embodiments, at least one clone-amplified sample nucleic acid molecule in the plurality of clonal amplified samples contains a single-stranded multimeric nucleic acid molecule, said specification 10 / 105 page 33 CN 121057626 A. The single-stranded multimeric nucleic acid molecule contains a repeating sequence of regularly occurring monomeric units. In some embodiments, the length of the single-stranded multimeric nucleic acid molecule is at least 10 kilobases. In some embodiments, at least one clone-amplified sample nucleic acid molecule in the plurality of clonal amplified samples further contains a double-stranded monomeric copy of said regularly occurring monomeric units. In some embodiments, the plurality of oligonucleotide molecules are present at various locations on the one or more surfaces with a substantially uniform surface density. In some embodiments, the plurality of oligonucleotide molecules are present at a local surface density of at least 100,000 molecules / µm² in a first region on one or more surfaces, and at a second local surface density in a second region on one or more surfaces. In some embodiments, the coating comprises: a first layer comprising monolayer polymer molecules tethered to one or more surfaces of the substrate; a second layer comprising a second monolayer polymer molecules tethered to the polymer molecules of the first layer; and a third layer comprising a third monolayer polymer molecules tethered to the polymer molecules of the second layer, wherein at least one of the first, second, or third layers comprises branched polymer molecules. In some embodiments, the third layer further comprises oligonucleotides tethered to the polymer molecules of the third layer. In some embodiments, the oligonucleotides tethered to the polymer molecules of the third layer are distributed at multiple depths throughout the third layer. In some embodiments, the coating further comprises: a fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer; and a fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer. In some embodiments, the polymer molecules of the fifth layer are further...The step includes an oligonucleotide tethered to the polymer molecules of the fifth layer. In some embodiments, the oligonucleotide tethered to the polymer molecules of the fifth layer is distributed throughout the fifth layer at multiple depths. In some embodiments, the at least one hydrophilic polymer coating comprises: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran. In some embodiments, when the cloned and amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine dye-3, the ratio of the fluorescence intensity of the cloned and amplified cyanine dye-3 labeled sample nucleic acid molecule or its complementary sequence to the fluorescence intensity of the nonspecific cyanine dye-3 adsorption background (B gap) shown in the images of the one or more surfaces is at least 3:1. In some embodiments, the ratio of the fluorescence intensity of the cloned and amplified cyanine dye-3 labeled sample nucleic acid molecule or its complementary sequence shown in the images of the one or more surfaces to the fluorescence intensity of the combination of the nonspecific cyanine dye-3 adsorption background and the nonspecific amplification background (B gap + B endoplasm) is at least 3:1. In some embodiments, when the cloned and amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine dye-3, the ratio of the fluorescence intensity of the cloned and amplified cyanine dye-3 labeled sample nucleic acid molecule or its complementary sequence to the fluorescence intensity of the nonspecific dye adsorption background (B gap) shown in the images of the one or more surfaces is at least 5:1. In some embodiments, the ratio of fluorescence intensity of the cloned, azocyanine-3-labeled sample nucleic acid molecules or their complementary sequences exhibited in the images of the one or more surfaces to the fluorescence intensity of the combination of nonspecific azocyanine-3 dye adsorption background and nonspecific amplification background (B interstitial + B endoplasm) is at least 5:1. In some embodiments, when the cloned, azocyanine-3-labeled sample nucleic acid molecules or their complementary sequences are used, the fluorescence images of the one or more surfaces acquired under non-signal saturation conditions using an inverted microscope equipped with a 20× objective lens, NA = 0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for azocyanine-3 emission, and a camera while the one or more surfaces are immersed in buffer, the fluorescence images exhibit a contrast-to-noise ratio (CNR) of at least 20. In some embodiments, the plurality of oligonucleotide molecules are described in the specification page 11 / 105, 34 CN.121057626 A is present at a surface density of at least 1,000 molecules / m². In some embodiments, the first reagent is configured to wet the one or more surfaces of the one or more channels. In some embodiments, the second reagent is configured to rewet the one or more surfaces of the one or more channels after at least partially drying the one or more surfaces through the air gap. In some embodiments, the flow cell system includes the flow cell device, wherein the flow cell system further includes: a fluid control device including: a first pump coupled to the outlet; and a dispenser configured to openly dispense one or more reagents to the inlet. In some embodiments, the first pump or the second pump is configured to introduce the air gap through the inlet and cause the air gap to flow at least partially through the one or more channels. In some embodiments, the air gap contains air. In some embodiments, the air gap contains dry air. In some embodiments, the air gap contains one or more inert gases. In some embodiments, the air gap contains one or more active gases. In some embodiments, the first reagent or the second reagent contains a liquid. In some embodiments, the first reagent or the second reagent lacks bubbles larger than a predetermined size. In some embodiments, the coating comprises a liquid repellent coating. In some embodiments, the coating comprises a superhydrophobic coating. In some embodiments, the coating comprises a smooth liquid-injected porous surface (SLIPS). In some embodiments, the coating comprises a smooth superhydrophobic covalently attached liquid (SOCAL) coating. In some embodiments, the coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates. In some embodiments, the coating is formed by impregnating a lubricant into one or more porous surfaces. In some embodiments, the lubricant comprises a liquid with a surface energy below about 20 mJ / m². In some embodiments, the lubricant comprises silicone oil. In some embodiments, the surface energy of the coating is below about 20 mJ / m². In some embodiments, the coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers. In some embodiments, the one or more brine monomers comprise dimethyldimethoxysilane. In some embodiments, the open landing region is fluidly connected to the one or more channels. In some embodiments, the open landing region is fluidly connected to one of the one or more channels. In some embodiments, the open landing region is located on the bottom substrate of the one or more substrates. In some embodiments, the inlet comprises a hole in the top substrate of the one or more substrates. In some embodiments, the...The aperture in the top substrate is positioned above at least a portion of the open landing area. In some embodiments, the dispenser is configured to openly dispense the one or more reagents through the aperture to the open landing area. In some embodiments, the dispenser is configured to openly dispense the one or more reagents from the tip of the dispenser into the open landing area. In some embodiments, the dispenser is configured to openly dispense the one or more reagents from the tip of the dispenser into the open landing area without a conduit between the dispenser and the open landing area. In some embodiments, at least a portion of the tip of the dispenser contacts the open landing area. In some embodiments, the tip of the dispenser does not contact the open landing area. In some embodiments, the flow cell device further includes a cleaning outlet in one or more substrates. In some embodiments, the cleaning outlet is fluidly connected to the inlet. In some embodiments, the cleaning outlet is fluidly connected to the open landing area. In some embodiments, the cleaning outlet is located in a top or bottom substrate of one or more substrates. In some embodiments, the cleaning outlet includes a side port on one or more substrates, wherein the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-direction; at least along a direction perpendicular to or nearly perpendicular to the y-direction; at least along a direction perpendicular to or nearly perpendicular to the z-direction; at least along a direction inclined to the x-direction; at least along a direction inclined to the y-direction; or at least along a direction inclined to the z-direction. In some embodiments, the cleaning outlet is configured to be coupled to the first pump or the second pump. In some embodiments, the one or more channels include microfluidic channels. In some embodiments, the one or more surfaces are coated with fluorescent beads, and the fluorescent beads are chemically immobilized to the one or more surfaces. In some embodiments, the fluorescent beads are covalently attached to the one or more surfaces. In some embodiments, the gap between the inner top surface and the inner bottom surface is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. In some embodiments, the height of the one or more channels is approximately 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. In some embodiments, the polynucleotides captured thereon are configured for imaging during a sequencing cycle. In some embodiments, the one or more substrates include a top substrate and a bottom substrate.Substrate. In some embodiments, the one or more channels are defined between the top substrate and the bottom substrate. In some embodiments, the one or more channels are at least partially defined in the top surface of the bottom substrate. In some embodiments, the one or more channels are at least partially defined in the bottom surface of the top substrate. In some embodiments, the one or more substrates further include an intermediate substrate. In some embodiments, the one or more channels are at least partially defined in the intermediate substrate. In some embodiments, the one or more substrates comprise glass or plastic. In some embodiments, at least a portion of the carrier is transparent. In some embodiments, at least a portion of the one or more substrates is transparent. In some embodiments, the carrier is solid-phase. In some embodiments, the one or more channels comprise 1, 2, 3, 4, 5, 6, 7, or 8 channels. In some embodiments, the one or more channels comprise 2, 4, 6, 8, or 10 channels. In some embodiments, the lane length of each of the one or more channels is less than about 70 mm, 75 mm, 80 mm, or 90 mm. In some embodiments, the lane width of each of the one or more channels is less than about 10 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, or 3 mm. In some embodiments, at least a portion of the open landing region includes a second coating comprising a smooth coating. In some embodiments, at least a portion of the open landing region includes a second coating comprising a liquid-repellent coating. In some embodiments, at least a portion of the open landing region includes a second coating comprising a superhydrophobic coating. In some embodiments, at least a portion of the open landing region includes a second coating comprising a smooth liquid-injected porous surface (SLIPS). In some embodiments, at least a portion of the open landing region includes a second coating comprising a smooth superhydrophobic covalently attached liquid (SOCAL) coating. In some embodiments, at least a portion of the open landing region includes a second coating comprising a liquid-like polymer brush surface covalently attached to the one or more substrates. In some embodiments, at least a portion of the open landing region includes a second coating comprising impregnating a lubricant in a porous surface to produce a coating with a surface energy below about 20 mJ / m². In some embodiments, at least a portion of the open landing region includes a second coating comprising an impregnating acid-catalyzed graft polycondensation of one or more brine monomers. In some embodiments, the one or more brine monomers comprise dimethyldimethoxysilane. In some embodiments, the use of theThe process of using the flow cell device includes removing at least a portion of the first reagent from at least a portion of the one or more channels during a DNA sequencing run. In some embodiments, the at least portion of the first reagent remains in the one or more channels during the DNA sequencing run. In some embodiments, the first reagent and the second reagent are different. In some embodiments, the at least portion of the one or more channels occupies more than 40% of the corresponding volume or length of each of the one or more channels. In some embodiments, the at least portion of the one or more channels occupies more than half of the corresponding volume or length of each of the one or more channels. In some embodiments, the at least portion of the one or more channels occupies more than 60% of the corresponding volume or length of each of the one or more channels. In some embodiments, the at least portion of the one or more channels occupies more than 70% of the corresponding volume or length of each of the one or more channels. In some embodiments, the at least portion of the one or more channels occupies more than 80% of the corresponding volume or length of each of the one or more channels. In some embodiments described on page 13 / 105 of CN 121057626 A, the process of using the flow cell device includes expelling residual amounts of the first reagent or the second reagent from the open landing area through a cleaning outlet of the flow cell device. In some embodiments, the clean outlet is configured to allow residual amounts of the first reagent on the open landing area to flow through the clean outlet. In some embodiments, the flow pool system includes the flow pool device, wherein the flow pool system further includes: a fluid control device including: a first pump fluidly connected to the clean outlet, wherein the first pump or a second pump is fluidly connected to the outlet; and a dispenser configured to openly dispense the one or more reagents to the inlet. In some embodiments, the first pump is configured to clean the open landing area by driving residual amounts of the first reagent away from the open landing area to flow through the clean outlet. In some embodiments, the process further includes: removing at least a portion of the first reagent from at least a portion of the one or more channels by driving fluids from the inlet through at least a portion of the one or more channels. In some embodiments, the residual amount of the first reagent on the open landing area comprises a meniscus of the first reagent. Brief Description of the Drawings

[0016] The novel features of the inventive concept are set forth in the appended claims. A better understanding of the features and advantages of the inventive concept will be obtained by referring to the following detailed description of illustrative embodiments, in which the principles of the inventive concept are utilized, and in the accompanying drawings:Figure 1 is a non-limiting example of a block diagram of a computer-implemented system for performing operations in DNA sequencing and sequencing analysis using the flow cell apparatus described herein, according to some embodiments.

[0017] Figure 2A is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0018] Figure 2B is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0019] Figure 2C is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0020] Figure 2D is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0021] Figure 2E is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0022] Figure 2F is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0023] Figure 2G is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0024] Figure 3A is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0025] FIG3B is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0026] FIG4A is a non-limiting example of a flow cell apparatus according to some embodiments.

[0027] FIG4B is a non-limiting example of a flow cell apparatus according to some embodiments.

[0028] FIG4C is a non-limiting example of a flow cell apparatus according to some embodiments.

[0029] FIG4D is a non-limiting example of a flow cell apparatus according to some embodiments.

[0030] FIG5A is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0031] FIG5B is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0032] FIG5C is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0033] FIG5D is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0034] FIG6A is a non-limiting example of a flow cell apparatus according to some embodiments.

[0035] FIG6B is a non-limiting example of a flow cell apparatus according to some embodiments.

[0036] FIG6C is a non-limiting example of a flow cell apparatus according to some embodiments. Specification 14 / 105 pages 37 CN 121057626 A

[0037] FIG7A is a schematic diagram illustrating a non-limiting example of a flow cell apparatus with an embedded tube according to some embodiments.

[0038] FIG7B is a schematic diagram illustrating a non-limiting example of a flow cell apparatus with an embedded tube according to some embodiments.

[0039] FIG7C is a schematic diagram illustrating a non-limiting example of a flow cell apparatus with an embedded tube according to some embodiments.

[0040] FIG7D is a schematic diagram illustrating a non-limiting example of a flow cell device with an embedded tube according to some embodiments.

[0041] FIG8 is a schematic diagram illustrating a non-limiting example of a flow cell device according to some embodiments.

[0042] FIG9A is a schematic diagram illustrating a non-limiting example of a flow cell device according to some embodiments.

[0043] FIG9B is a non-limiting example of a flow cell device according to some embodiments.

[0044] FIG9C is a non-limiting example of a flow cell device according to some embodiments.

[0045] FIG9D is a non-limiting example of a flow cell device according to some embodiments.

[0046] FIG9E is a non-limiting example of a flow cell device according to some embodiments.

[0047] FIG10 is a schematic diagram illustrating a non-limiting example of a flow cell device according to some embodiments.

[0048] FIG11 is a schematic diagram illustrating a non-limiting example of a flow cell device according to some embodiments.

[0049] FIG12A is a non-limiting example of a flow cell device according to some embodiments.

[0050] FIG12B is a non-limiting example of a flow cell device according to some embodiments.

[0051] FIG12C is a non-limiting example of a flow cell apparatus according to some embodiments.

[0052] FIG12D is a non-limiting example of a flow cell apparatus according to some embodiments.

[0053] FIG12E is a non-limiting example of a flow cell apparatus according to some embodiments.

[0054] FIG12F is a non-limiting example of a flow cell apparatus according to some embodiments.

[0055] FIG12G is a non-limiting example of a flow cell apparatus according to some embodiments.

[0056] FIG12H is a non-limiting example of a flow cell apparatus according to some embodiments.

[0057] FIG13 is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0058] FIG14A is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0059] FIG14B is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0060] FIG14C is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0061] FIG14D is a schematic diagram illustrating a non-limiting example of a flow cell apparatus according to some embodiments.

[0062] FIG15 is a schematic diagram illustrating a non-limiting example of a linear single-stranded library molecule.

[0063] FIG16 is a schematic diagram illustrating a non-limiting example of a linear single-stranded library molecule.

[0064] FIG17 is a schematic diagram illustrating various configurations of multivalent molecules.

[0065] FIG18 is a schematic diagram illustrating a non-limiting example of a multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0066] FIG19 is a schematic diagram illustrating a non-limiting example of a dendritic macromolecular core attached to multiple nucleotide arms.

[0067] FIG20 is a schematic diagram of a non-limiting example of a multivalent molecule comprising a nucleus attached to multiple nucleotide arms, wherein the nucleotide arms comprise biotin, spacers, linkers, and nucleotide units.

[0068] FIG21 is a schematic diagram of a non-limiting example of a nucleotide arm comprising a nucleus attachment portion, spacers, linkers, and nucleotide units.

[0069] FIG22 is a schematic diagram of the chemical structure of a spacer (top) and various linkers, as shown on page 15 / 105 of the specification, CN 121057626 A, comprising 11-atom linkers, 16-atom linkers, 23-atom linkers, and N3 linkers (bottom).

[0070] FIG23 is a schematic diagram of a non-limiting example of the chemical structure of various linkers comprising linkers 1-9.

[0071] FIG24 is a schematic diagram of a non-limiting example of the chemical structure of various linkers that are attached to / attached to nucleotide units.

[0072] FIG25 is a schematic diagram of a non-limiting example of the chemical structure of various linkers that are attached to / attached to nucleotide units.

[0073] FIG26 is a schematic diagram of a non-limiting example of the chemical structure of various connectors that are attached to / attached to nucleotide units.

[0074] FIG27 is a schematic diagram of a non-limiting example of the chemical structure of various connectors that are attached to / attached to nucleotide units.

[0075] FIG28 is a schematic diagram of a non-limiting example of the chemical structure of a biotinylated nucleotide arm.

[0076] FIG29 is a schematic diagram of a non-limiting example of a flow cell device.

[0077] FIG30 is a schematic diagram of a non-limiting example of a flow cell system according to some embodiments.

[0078] FIG31 is a schematic diagram of a non-limiting example of a flow cell device according to some embodiments.

[0079] FIG32 is a schematic diagram of a non-limiting example of a flow cell device according to some embodiments.

[0080] FIG33A-33F are non-limiting examples of flow cell devices according to some embodiments. FIG33A is a perspective view of a substrate of an embodiment of the flow cell device disclosed herein. FIG33B is a top view of the flow cell device in FIG33A. Figure 33C is a cross-sectional view of the flow cell apparatus at point D-D' in Figure 33B. Figure 33D is a perspective view of the substrate of another embodiment of the flow cell apparatus disclosed herein. Figure 33E is a perspective view of the substrate of yet another embodiment of the flow cell apparatus disclosed herein. Figure 33F shows a perspective view and a top view of yet another embodiment of the flow cell apparatus disclosed herein.

[0081] Figures 34A-34C are non-limiting examples of fluid control devices for delivering reagents to a flow cell system according to some embodiments. Figure 34A shows a fluid control device including a dispenser (680a) and a continuous track (691a). Figure 34B shows a fluid control device including a dispensing plate (692a) having an electrowetting surface. Figure 34C shows...A fluid control device comprising a reagent reservoir (694a) and a pipette (693a).

[0082] FIG35 is a graph showing the level of contamination achieved by the flow cell system disclosed herein compared to existing flow cell systems.

[0083] FIG36 is a non-limiting example of a block diagram of a computer system for fluid control and for performing sequencing and sequencing analysis according to some embodiments.

[0084] FIG37A-37E are non-limiting examples of the flow cell apparatus in FIG33A-33D. FIG37A is a perspective view of the flow cell apparatus. FIG37B is a perspective view of the flow cell apparatus showing a top substrate, a middle substrate, and a bottom substrate. FIG37C is a top view of the top substrate of the flow cell apparatus. FIG37D is a top view of the middle substrate of the flow cell apparatus. FIG37E is a top view of the bottom substrate of the flow cell apparatus.

[0085] FIG38A-38E are non-limiting examples of the flow cell apparatus disclosed herein. FIG38A is a perspective view of the flow cell apparatus. Figure 38B is a perspective view of a flow cell apparatus showing a top substrate, a middle substrate, and a bottom substrate. Figure 38C is a top view of the top substrate of the flow cell apparatus. Figure 38D is a top view of the middle substrate of the flow cell apparatus. Figure 38E is a top view of the bottom substrate of the flow cell apparatus.

[0086] Figures 39A-39C are non-limiting examples of the flow cell apparatus disclosed herein. Figure 39A is a top view of an embodiment of the flow cell apparatus. Figure 39B is a top view of another embodiment of the flow cell apparatus. Figure 39C is a top view of yet another embodiment of the flow cell apparatus. Specification 16 / 105 pages 39 CN 121057626 A

[0087] Figures 40A-40G are non-limiting examples of the flow cell apparatus disclosed herein. Figure 40A is a side view of the flow cell apparatus. Figure 40B shows a cross-sectional view along line A-A in Figure 40A. Figure 40C is a top view of the flow cell apparatus. Figure 40D is a cross-sectional view along line B-B in Figure 40B. Figure 40E shows an extended view of region A in Figure 40B. Figure 40F shows an extended view of region C in Figure 40C. Figure 40G shows an extended view of region B in Figure 40D.

[0088] Figures 41A-41C are embodiments of the flow cell apparatus of Figure 33E, including top (Figure 41A), perspective (Figure 41B), and perspective (Figure 41C) views of the bottom substrate, middle substrate, and top substrate.

[0089] Figure 42 is a graph showing the contamination level of a single patch and the average contamination level across multiple patches of the flow cell apparatus implemented by the flow cell system disclosed herein.

[0090] Figure 43A is a non-limiting example of an embodiment of a flow cell apparatus with a filter. In this specific embodiment, the filter reduces or eliminates contaminants that may enter the channel from the open landing area.

[0091] Figure 43B is a non-limiting example of an embodiment of a flow cell apparatus with a filter. In this specific embodiment, the filter reduces or eliminates contaminants that may enter the channel from the open landing area. Detailed Description

[0092] This document describes systems and apparatuses for analyzing different nucleic acid sequences, such as nucleic acid sequences from amplified nucleic acid arrays in a flow cell or from immobilized nucleic acid arrays. The systems and apparatuses described herein can also be used, for example, for sequencing in comparative genomics, tracking gene expression, microRNA sequence analysis, epigenomics, and aptamer and phage display library characterization, as well as other sequencing applications. The systems and apparatuses described herein encompass various combinations of optical, mechanical, fluidic, thermal, electrical, and computational devices / aspects.

[0093] The advantages of the disclosed flow cell apparatus, fluid control device, and system include, but are not limited to: significantly reduced consumable costs (e.g., compared to currently available nucleic acid sequencing systems); efficient and effective cleaning of the flow cell apparatus, thereby reducing contamination during sequencing, such as contamination from residual reagents; shortened reagent delivery time, reduced washing time, and improved reagent homogeneity on the flow cell; reduced apparatus and system manufacturing / maintenance complexity and cost; flexible system throughput, and flexible adaptability of the system to different sequencing applications.

[0094] The advantages of the disclosed flow cell apparatus and system may also include: reduced bending stress on the flow cell substrate, which may result in improved optical flatness and performance; improved thermal contact between the flow cell and the thermal control device, which may improve the chemical properties of the flow cell; improved spatial clearance for distributing open fluid to the flow cell apparatus; increased surface area for colony development and imaging, which may increase throughput for sequencing applications; and improved fluid interface sealing compared to existing flow cell apparatuses.

[0095] Some of the design features of the disclosed flow cell devices, cartridges, and systems include, but are not limited to: open dispensing tips in the fluid control device and open landing areas on the flow cell device to allow open delivery of reagents and / or washing buffers without the complexity and cost of existing piping, and to make the system flexible to adapt to different sequencing applications; a smooth coating that facilitates fluid transfer and residual cleaning from the open landing area; a clean outlet fluidly connected to the open landing area to facilitate cleaning of liquid menisci that cannot be effectively cleaned using washing reagents or washing buffers alone; positioning of the clean outlet that allows for effective cleaning during the sequencing process and facilitates expansion of the flow cell device to increase sequencing throughput without replacing the clean outlet; a channel coating that allows for the removal of air gaps between two fluid reagents without impairing subsequent sequencing reactions; and compatibility with a variety of detection methods such as fluorescence imaging.

[0096] Although the disclosed flow cell devices and systems are described primarily in the context of their use in nucleic acid sequencing applications.However, the various aspects of the disclosed apparatus and systems can be used not only for nucleic acid sequencing but also for any other type of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications as described in this specification (pages 17 / 105, CN 121057626 A). It should be understood that the different aspects of the disclosed apparatus and systems can be understood individually, jointly, or in combination with each other.

[0097] Sequencing Systems In some embodiments, flow cell apparatuses and systems are disclosed herein that can be used to perform or facilitate DNA sequencing analysis using sequencing systems, such as next-generation sequencing (NGS) systems. Sequencing systems can utilize a variety of sequencing technologies, including but not limited to the sequencing technologies disclosed herein.

[0098] Figure 1 shows a block diagram of a computer-implemented system 100 for performing sequencing and sequencing analysis according to one or more embodiments disclosed herein. System 100 has a sequencing system 110 that includes a flow cell apparatus 112, a sequencer 114, an imager 116, a data storage device 122, and a user interface 124. Sequencing system 110 can be connected to cloud 130. Sequencing system 110 may include one or more of the following: dedicated processor 118, field-programmable gate array (FPGA) 120, and computer system 126.

[0099] In some embodiments, flow cell device 112 is configured to capture DNA fragments and form DNA sequences for base calling on the flow cell device. Flow cell device 112 may include a vector as disclosed herein. The vector may be a solid-phase vector. As disclosed herein, the vector may include a surface coating thereon. The surface coating may be a polymer coating as disclosed herein. The surface coating may be on the surface of one or more channels of the flow cell device. Different or identical surfaces may be placed on the surface of the inlet of the flow cell device.

[0100] Flow cell device 112 may include a plurality of patches or imaging regions thereon, and each patch may be divided into a sub-pattern grid. Each sub-pattern may include a plurality of clusters or communities thereon. As a non-limiting example, the flow cell may have 424 patches, and each patch may be divided into a 6 x 9 grid, thus having 54 sub-patterns. A flow cell image as disclosed herein may be an image of signals comprising multiple clusters or communities. A flow cell image may include one or more signal patches or one or more signal sub-patterns. In some embodiments, a flow cell image may be an image including all patches and approximately all signals thereon. Flow cell images may be acquired from channels using imager 116 during an imaging or sequencing cycle. In some embodiments, each patch may include millions of communities or clusters. As a non-limiting example, a patch may include approximately one to ten million clusters or communities. Each community may be a collection of many copies of DNA fragments.

[0101] Further details of the flow cell apparatus 112 and its functional and structural elements are disclosed herein in conjunction with figures, such as Figures 2A-2G, 3A-3B, 4-14, 30-32, 33A-33F, 34A-34C, and 35.

[0102] The sequencer 114 may be configured to flow a mixture of nucleotides onto the flow cell apparatus 112, cleave inhibitors from the nucleotides between flow steps, and perform other steps for forming a DNA sequence on the flow cell apparatus 112. The nucleotides may have attached fluorescent elements that emit light or energy at wavelengths indicating the type of nucleotide. Each type of fluorescent element may correspond to a specific nucleotide base (e.g., A, G, C, T). The fluorescent elements may emit light at visible wavelengths. In some embodiments, the sequencer 114 and the flow cell apparatus 112 may be configured to perform various sequencing methods disclosed herein, such as affinity sequencing or sequencing-by-synthesis.

[0103] For example, each nucleotide base may be assigned a color. Different types of nucleotides may have different colors. For example, adenine (A) can be red, cytosine (C) can be blue, guanine (G) can be green, and thymine (T) can be yellow. The color or wavelength of the fluorescent element for each nucleotide can be selected so that the nucleotides can be distinguished from each other based on the wavelength of light emitted by the fluorescent element.

[0104] Imager 116 can be configured to capture an image of flow cell apparatus 112 after each flow step. In one embodiment, imager 116 is a camera, such as a CMOS or CCD camera, configured to capture digital images. The camera can be configured to capture an image of the wavelength of the fluorescent element that binds to the nucleotide. The image can be referred to as a flow cell image. Specification 18 / 105 pages 41 CN 121057626 A

[0105] In some embodiments, imager 116 may include one or more optical systems disclosed herein. The optical system can be configured to capture optical signals from the flow cell and generate a corresponding digital image. The digital image can then be used for base calling.

[0106] In one embodiment, images of the flow cell can be captured in groups, wherein each image in the group is taken at a wavelength or spectrum that matches or includes one of the fluorescent elements. In another embodiment, the images can be captured as a single image capturing all wavelengths of the fluorescent elements.

[0107] The resolution of the imager 116 controls the level of detail in the flow cell image, including pixel size. This resolution is critical in existing systems because of the accuracy of their control point finding algorithms in identifying community centers. In some embodiments, the image resolution of the flow cell images disclosed herein can be from about 10 nanometers (nm) to hundreds of nm or higher.One way to improve the accuracy of the search is to improve the resolution of the imager 116 or to improve the processing of the images captured by the imager 116. Detection of community centers in pixels other than those detected by the point search algorithm can be performed. These methods can allow for improved accuracy in community center detection without increasing the resolution of the imager 116. The resolution of the imager can even be lower than that of existing systems with comparable performance, which can reduce the cost of the sequencing system 110.

[0108] Image quality of the flow cell image controls the base call quality. One way to improve the accuracy of base call is to improve the imager 116 or to improve the processing of the images captured by the imager 116 to obtain better image quality.

[0109] After performing the base call, the sequencing read can be output from the system to the cloud 130 or computer system 126, depending on the choice of performing some processing on the base call result. The sequencing read herein can be a forward read (R1), a reverse read (R2), or both. The sequencing read herein can be any ordered sequence of A, T, C, and G bases.

[0110] In some embodiments, sequencing reads can be directly transmitted to computer system 126 for subsequent analyses such as adapter trimming.

[0111] These sequencing analysis methods, including preliminary analysis, secondary analysis, or combinations thereof, can advantageously be executed in parallel within computer system 126 without interfering with or delaying the existing sequencing workflow of system 100. The results of the sequencing analysis can be used to generate sequencing results for the user. Some or all of the operations of the sequencing process can advantageously be performed by an FPGA, and data can be transferred between the CPU and the FPGA to reduce the total operation time of methods that do not use FPGA operations.

[0112] The operations or actions disclosed herein can be performed by a dedicated processor 118, FPGA 120, computer system 126, or a combination thereof. One or more operations or actions in the methods disclosed herein can be performed by a dedicated processor 118, FPGA 120, computer system 126, or a combination thereof. In some embodiments, which operations or actions will be performed by the dedicated processor 118, FPGA 120, computer system 126, or a combination thereof may be determined based on one or more of the following: computation time for a particular operation, computational complexity in a particular operation, the need for data transfer between hardware devices, or a combination thereof.

[0113] The computer system 126 may include one or more general-purpose computers or computer processors that provide an interface for running various programs in an operating system such as Windows™ or Linux™. Such operating systems can provide users with great flexibility.

[0114] In some embodiments, the computer processor may control a flow pool system or flow pool device as disclosed herein.Various structural elements of the device. For example, a computer processor can execute computer instructions to control a force-applying mechanism that applies force or pressure to a connector, manifold, seal, or combination thereof to achieve a sealed fluid connection with the flow cell device.

[0115] The dedicated processor 118 may not be a general-purpose processor, but a custom processor with specific hardware or instructions for performing method steps. In some embodiments, the dedicated processor may include various processing units. In some embodiments, the dedicated processor may include: an application-specific integrated circuit (ASIC) chip, a neural processing unit (NPU), an artificial intelligence chip specification 19 / 105 pages 42 CN 121057626 A (AI chip), a tensor processing unit (TPU), and a graphics processing unit (GPU). The dedicated processor may include integrated circuits that may be reconfigurable or non-configurable, but optimized for specific computational tasks, such as using neural networks for prediction. The dedicated processor can run specific software directly without an operating system. The lack of an operating system reduces overhead, but at the cost of the flexibility that the processor can execute. The dedicated processor may use a custom programming language that can be designed to operate more efficiently than software running on a general-purpose computer. This can increase the speed of execution steps and allow for real-time processing.

[0116] In some embodiments, the FPGA 120 can be configured to perform the operations of the sequencing analysis methods described herein. The FPGA is programmed as hardware capable of performing specific tasks. Software steps can be translated into hardware components using a special programming language. Once the FPGA is programmed, the hardware directly processes the digital data provided to it without running software. Instead, the FPGA uses logic gates and registers to process the digital data. Since the operating system does not require any overhead, the FPGA can process data faster than a general-purpose computer. Similar to dedicated processors, this comes at the cost of flexibility.

[0117] The lack of software overhead can also allow the FPGA to operate faster than a general-purpose processor, such as a CPU, although this will depend on the exact processing to be performed and the specific FPGA and processor.

[0118] A group of FPGAs 120 can be configured to perform these steps in parallel. For example, many FPGAs 120 can be configured to perform processing steps targeting selected regions in an image, a set of images, a sub-plot, or one or more images. Each FPGA 120 can execute its own processing step portion simultaneously, thereby reducing the time required to process data. This can allow processing steps to be completed in real time. The following provides a further discussion of the use of FPGAs.

[0119] Real-time execution of processing steps allows the system to use less storage because data can be processed as it is received. This is an improvement over existing systems that may require storing data before processing it, and the latter can...More storage or access may be required for the computer system located in cloud 130.

[0120] In some embodiments, data storage device 122 is used to store information for sequencing analysis or obtained from sequencing analysis. For example, DNA sequences determined after adaptor trimming may be stored in data storage device 122. Compressed or uncompressed sequencing data or a combination thereof may be stored in data storage device 122. FASTQ files may also be stored in data storage device 122.

[0121] User interface 124 may be used by a user to operate the sequencing system or access data stored in data storage device 122 or computer system 126.

[0122] Computer system 126 may control the general operation of the sequencing system and may be connected to user interface 124. Computer system 126 may also perform sequencing analysis steps such as image registration, color correction, base calling, adaptor trimming, demultiplexing, etc. In some embodiments, computer system 126 is computer system 800, as described in more detail in FIG8. Computer system 126 may store information about the operation of sequencing system 110, such as configuration information, instructions for operating sequencing system 110, or user information. Computer system 126 can be configured to transfer information between sequencing system 110 and cloud 130.

[0123] As discussed above, sequencing system 110 may have a dedicated processor 118, FPGA 120, or computer system 126. The sequencing system may use one, two, or all of these elements to perform the processing described above. In some embodiments, when these elements are present together, the processing tasks are separated therebetween. For example, FPGA 120 may be used to perform some or all of the sequencing analysis operations, and optionally, dedicated processor 118 may be used to perform other parts of the sequencing analysis, such as predicting the community location of in situ samples. Computer system 126 may perform other processing functions for sequencing system 110. Various combinations of these elements may allow for various system embodiments that balance the efficiency and speed of processing with the cost of the processing elements.

[0124] Cloud 130 may be a network, a remote storage device, or some other remote computing system separate from sequencing system 110. The connection to the cloud 130 can allow access to data stored outside the sequencing system 110 or allow updates to the software within the sequencing system 110.

[0125] Flow cell apparatus In some embodiments, flow cell apparatuses and systems are disclosed herein that can be used to perform or facilitate DNA sequencing analysis. The flow cell apparatus described herein can be used to immobilize template nucleic acid molecules derived from biological samples and introduce repeated flows of sequencing reagents (e.g., sequencing-by-binding, sequencing-by-synthesis, or affinity sequencing or thereof).(Combination) to attach labeled nucleotides to specific locations in a template sequence. A series of labeled signals are detected and decoded to reveal the nucleotide sequence of the template molecule, such as a fixed or amplified nucleic acid template molecule or a combination thereof attached to the surface of the flow cell.

[0126] In some embodiments, the sample herein may be a conventional 2D DNA sequencing sample. In some embodiments, the sample herein may be a 3D volume sample, such as an in situ sample of cells or tissue.

[0127] The flow cell device may include a carrier comprising one or more substrates; one or more channels defined by the one or more substrates and extending along a first direction, along the y-axis and between an inlet and an outlet; and one or more seals positioned on the one or more substrates to improve the sealing of fluid communication between the flow cell device and any other component of the flow control device or sequencing system. In some embodiments, the one or more channels are in fluid communication with a fluid passage of a manifold or connector. Such fluid communication between the channel and the fluid passage may be direct or indirect via a clean outlet. An open landing area may also be in fluid communication with a fluid passage of a manifold or connector, either directly or via a clean outlet. The fluid passage of the manifold or connector can then be in fluid communication with a fluid control device such as a pump or vacuum device, thereby allowing reagents to be removed from the open landing area and / or channels. The fluid passage can also be used to introduce liquids or gases into the open landing area or the one or more channels when needed.

[0128] In some embodiments, the flow cell device 112 disclosed herein may include a carrier 210 having one or more substrates, multiple channels, an inlet, a clean outlet, and an outlet. Figures 2-14 show a non-limiting example of the flow cell device 112.

[0129] In some embodiments, the flow cell device 112 disclosed herein may include a carrier 210. The carrier may be solid. At least a portion of the carrier 210 may be transparent, such that light transmitted from a light source from an imager (116 in Figure 1) can travel through the transparent portion of the carrier and reach the sample located on the flow cell device 112.

[0130] The carrier 210 may include one or more substrates 220, 230.

[0131] As shown in Figures 2A-2B, the one or more substrates may include a top substrate 220 and a bottom substrate 230. When the flow cell device 112 is placed in the sequencing system 110 for imaging, the top substrate 220 may be closer to the camera of the imager 116 along the z-axis direction than the bottom substrate 230. The bottom substrate 230 may be closer to the translation stage of the sequencing system 110 than the top substrate 220 to fix and support the flow cell device 112 during sequencing. The z-direction may be orthogonal to the image plane.In some embodiments, the top and / or bottom substrates may include one or more layers. For example, the top substrate may include a second, third, or even more layers 221, and the bottom substrate may include one or more layers 231, said one or more layers being mechanically fixed together (e.g., glued or attached with adhesive), with at least some overlapping areas in the x-y plane. 227 represents thickness a, and 228 represents thickness b, as can be seen in FIG2A.

[0132] In some embodiments, the flow cell device 112 may further include an intermediate substrate located between the top substrate and the bottom substrate.

[0133] The top substrate (including 220 and 221) may include a first thickness above the first portion 253 of the channel and a second thickness around the second portion 254 of the channel 250. The second thickness may be greater than the first thickness. In some embodiments, the second thickness may be 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the first thickness.

[0134] The bottom substrate (including 230 and 231) may include a third thickness above the first portion 253 of the channel and a fourth thickness above the second portion 254 of the channel. The fourth thickness may be greater than the third thickness. The fourth thickness may be 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the third thickness. The thickness herein may be along the z-direction.

[0135] In some embodiments, the second thickness may be the same as the first thickness. The fourth layer thickness may be the same as or greater than the third layer thickness. In other words, the top substrate and / or the bottom substrate may have a uniform thickness along the y-axis from one end of the flow cell device to the other end. Having a uniform thickness in the top substrate may advantageously facilitate homogeneous light transmission from the light source to the sample fixed on the flow cell device.

[0136] In some embodiments, the one or more substrates may include two, three, four, five, six, or even more substrates. In some embodiments, when the one or more substrates are assembled together to form a flow cell device, one, two, three, four or more surfaces may be formed on which a sample may be fixed. For example, the one or more substrates may form two surfaces, such as a top surface and a bottom surface of a fluid channel displaced relative to each other along the z-axis. As another example, the one or more substrates may form four surfaces, such as a top surface and a bottom surface of a first fluid channel displaced relative to each other along the z-axis, and a top surface and a bottom surface of a second fluid channel displaced relative to each other along the z-axis, wherein the first fluid channel and the second fluid channel are located at different z-positions along the z-axis.

[0137] In some embodiments, the flow cell device 112 disclosed herein may comprise having one or more substratesCarrier, multiple channels, inlet and outlet. Figures 30-32 and 33A-33F illustrate further embodiments of the flow cell apparatus.

[0138] In some embodiments, the flow cell apparatus 112 disclosed herein may include carriers 210, 510a. Carriers 210, 510a may be solid. At least a portion of carriers 210, 510a may be transparent, such that light transmitted from a light source from an imager (116 in FIG. 1) can travel through the transparent portion of the carrier and reach the sample located on the flow cell apparatus 112.

[0139] Carriers 210, 510a may include one or more substrates 220, 230, 320a, 322a, 330a, 520a, 420a, 422a, 430a, 522a, 530a, 722a. As shown in Figures 31-32, the one or more substrates may include top substrates 320a, 420a and bottom substrates 330a, 430a. When the flow cell device 112 is placed in the sequencing system 110 for imaging, the top substrates 320a and 420a can be closer to the camera of the imager 116 along the z-axis direction than the bottom substrates 330a and 430a. The bottom substrates 330a and 430a can be closer to the translation stage of the sequencing system 110 than the top substrates 320a and 420a to fix and support the flow cell device 112 during sequencing.

[0140] In some embodiments, the flow cell device 112 may further include intermediate substrates 322a, 422a, 522a, and 722a between the top substrates 320a, 420a, and 520a and the bottom substrates 330a, 430a, and 530a, as shown in Figures 31-32, 33A, and 33C.

[0141] Each substrate may have a predetermined thickness, and different substrates may have different thicknesses. In some embodiments, each substrate may have a uniform thickness along the z-direction. In some embodiments, each substrate may have a uniform thickness along the z-direction in at least a portion of the substrate (e.g., in a first portion 225 or a second portion 235). In some embodiments, the portion having a uniform thickness may cover a channel or imaging region of the flow cell device 112.

[0142] In some embodiments, the thickness of the top or bottom substrate may be from about 0.2 mm to about 5 mm. In some embodiments, the thickness of the top or bottom substrate may be from about 0.6 mm to about 3 mm. In some embodiments, the thickness of the top or bottom substrate may be from about 0.8 mm to about 2 mm. In some embodiments, the thickness of the top or bottom substrate may be from about 0.8 mm to about 1.5 mm. In some embodiments, the thickness of the top or bottom substrate may be from about 0.8 mm to about 1.2 mm. In some embodiments, the thickness of the top or bottom substrate may be from about 0.9 mm to about 1.1 mm. (Page 22 / 105 of the specification)45 CN 121057626 A

[0143] In some embodiments, the thickness of the top or bottom substrate may be from 0.2 mm to 5 mm. In some embodiments, the thickness of the top or bottom substrate may be from 0.6 mm to 3 mm. In some embodiments, the thickness of the top or bottom substrate may be from 0.8 mm to 2 mm. In some embodiments, the thickness of the top or bottom substrate may be from 0.8 mm to 1.5 mm. In some embodiments, the thickness of the top or bottom substrate may be from 0.8 mm to about 1.2 mm. In some embodiments, the thickness of the top or bottom substrate may be from 0.9 mm to 1.1 mm. In some embodiments, the thickness of the top or bottom substrate may be from 0.95 mm to 1.05 mm.

[0144] In some embodiments, the thickness of the intermediate substrate may be from about 40 μm to 200 μm. In some embodiments, the thickness of the intermediate substrate may be from about 40 μm to 150 μm. In some embodiments, the thickness of the intermediate substrate may be from about 40 μm to 70 μm. In some embodiments, the thickness of the intermediate substrate may be from about 80 μm to 120 μm. In some embodiments, the thickness of the intermediate substrate can be approximately 60 μm to 90 μm.

[0145] In some embodiments, the thickness of the intermediate substrate can be 40 μm to 200 μm. In some embodiments, the thickness of the intermediate substrate can be 40 μm to 150 μm. In some embodiments, the thickness of the intermediate substrate can be 40 μm to 70 μm. In some embodiments, the thickness of the intermediate substrate can be 80 μm to 120 μm. In some embodiments, the thickness of the intermediate substrate can be 60 μm to 90 μm.

[0146] In some embodiments, the thickness a of the flow cell device 112 can be in the range of 1 mm to 5 mm. In some embodiments, the thickness a of the flow cell device 112 can be in the range of 1.5 mm to 3.5 mm. In some embodiments, the thickness of the flow cell device 112 can be in the range of 2 mm to 6 mm.

[0147] In some embodiments, the substrate can have an elongated shape extending along the y-axis. In some embodiments, the substrate can have various shapes, such as rectangular, square, etc.

[0148] In some embodiments, the one or more substrates can have one or more planar surfaces. In some embodiments, the one or more substrates do not contain any curvature perceptible to the naked eye, for example, as shown in Figures 2A, 30-32, such that the one or more substrates can have a planar surface. In some embodiments, the flatness of the substrate surface can be measured as the height from its peak to its valley in a direction orthogonal to the surface. For example, along a direction orthogonal to the surface, the height can be less than about 0.01 mm, 0.02 mm, 0.03 mm, or 0.04 mm.The flatness of the substrate can be 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm. In other words, the flat surface of the substrate can fit between two parallel 2D planes that are less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm apart. In some embodiments, the flatness of the substrate surface can include a height of less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm from peak to valley. However, in some embodiments, the substrate does not have to be planar. Alternatively, part or all of one or more substrates may be curved. In some embodiments, the flatness of the surface of the substrate from peak to valley may be less than about 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm. In some embodiments, the flatness of the surface of the substrate from peak to valley may be less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm. However, in some embodiments, the substrate need not be planar. Alternatively, part or all of one or more substrates may be curved. As an example, surfaces A, B, and C in Figures 40A-40G may have a flatness of about 0.02 mm or 0.03 mm from peak to valley.

[0149] In some embodiments, the carrier or the one or more substrates may comprise glass or plastic. In some embodiments, the carrier or the one or more substrates are all glass, all plastic, or a combination of glass and plastic. Figure 2B shows one or more layers 231 of a bottom substrate, which may include a bonded plastic at the second portion 236. In some embodiments, the carrier or one or more substrates may include tape, such as pressure-sensitive adhesive (PSA) tape. In some embodiments, the carrier or one or more substrates are all-glass or all-plastic. For example, the intermediate substrate shown in Figure 31 can be made of PSA tape and can be conveniently fixed to the top substrate and the bottom substrate.

[0150] In some embodiments, the substrate may define one or more channels of the flow pool device 112. In some embodiments, the channel 250 may allow fluid, such as liquid or gas, to flow through it. In some embodiments, substrate 320a,322a, 330a, 420a, 422a, 430a, 520a, 522a and / or 530a may define one or more channels 250, 350a, 450a, 550a of the flow cell device 112. In some embodiments, channels 250, 350a, 450a, 550a may allow fluid, such as liquid or gas, to flow through them.

[0151] The gas referred to herein may comprise one type of gas or a combination of different types of gases. In some embodiments, the gas comprises air. The gas may comprise dry air. In some embodiments, the gas comprises one or more inert gases. In some embodiments, the gas comprises one or more active gases.

[0152] The reagent referred to herein may comprise a liquid. In some embodiments, the reagent does not contain bubbles larger than a predetermined size. In some embodiments, a first reagent is configured to wet a first coating on the surface of the one or more channels. In some embodiments, a second reagent is configured to rewet the surface of the one or more channels after the surface has been at least partially dried by air gaps.

[0153] In some embodiments, a first portion of channel 253 in one or more channels 250 includes a first z-position, and a second portion 254 of the channel includes a second z-position different from the first z-position, for example, as shown in FIG2A.

[0154] In some embodiments, the channel may be defined by a top inner surface 251 and a bottom inner surface 252 of a substrate. Specifically, channel 250 may each include a lumen defined by a top inner surface 251 and a bottom inner surface 252 of a substrate surrounding a lumen. In some embodiments, the channel includes a first portion located at the first z-position and a second portion located at a second z-position offset from the first z-position, as shown in FIG2A-2B. The top inner surface 251 and the bottom inner surface 252 may extend from a cleaning outlet 270 and / or an inlet 240 into the channel, and then into an outlet 260.

[0155] In some embodiments, channels 250a, 350a, 450a, and 550a may be defined by a top inner surface 521a and a bottom inner surface 521a of a substrate. Specifically, channels 250a and 550a may each include a cavity 551a defined by a top inner surface 521a and a bottom inner surface 521a of a substrate surrounding a cavity 551a, and a groove in the top surface, bottom surface, or both, without an intermediate substrate.

[0156] In some embodiments, the one or more substrates may include two, three, four, five, six, or even more substrates. In some embodiments, when the one or more substrates are assembled together to form a flow cell device, one, two, three, four, or more surfaces may be formed on which a sample may be fixed. For example, the one or more substrates may form two surfaces, such as a top surface and a bottom surface of a fluid channel that are displaced relative to each other along the z-axis. As anotherIn one example, the one or more substrates may form four surfaces, such as a top surface and a bottom surface of a first fluid channel that are displaced relative to each other along the z-axis, and a top surface and a bottom surface of a second fluid channel that are displaced relative to each other along the z-axis, wherein the first fluid channel and the second fluid channel are located at different z-positions along the z-axis.

[0157] In some embodiments, channels 350a, 450a, 550a may be defined by a top substrate and a bottom substrate plus an intermediate substrate 322a, 422a, 522a. The intermediate substrate may include a gap extending along the longitudinal axis or y-axis of the intermediate substrate, such as an elongated gap. The width of the gap may define the width of channels 350a, 450a, 550a along the x-axis, and the length of the gap along the y-direction may define the length of the channel. Figures 31-32 and 33A illustrate a flow cell device having channels 350a, 450a, 550a defined by a top substrate, an intermediate substrate, and a bottom substrate.

[0158] In some embodiments, the channels are microfluidic channels. In some embodiments, the gap or height between the top inner surface and the bottom inner surface of the substrate defining the channel along the z-direction is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. In some embodiments, the gap or height of the channel does not exceed about 100 μm. In some embodiments, the gap or height of the channel does not exceed about 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm.

[0159] In some embodiments, the gap or height between the top inner surface 251 and the bottom inner surface 252 of the substrate defining the channel along the z-direction is 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. In some embodiments, the gap or height of the channel does not exceed 100 μm. In some embodiments, the gap or height of the channel does not exceed 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm.

[0160] In some embodiments, the length of the channel along the y-direction is approximately 120 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, or 30 mm. In some embodiments, the length of the channel does not exceed approximately 100 mm. In some embodiments, the length of the channel does not exceed about 80 mm, 75 mm, 70 mm, 65 mm, 60 mm, 55 mm, 50 mm, 45 mm, or 40 mm.

[0161] In some embodiments, the length of the channel along the y-direction is 120 mm, 100 mm, or 120 mm.The channel width is approximately 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 8 mm, or 5 mm. In some embodiments, the channel length does not exceed 100 mm. In some embodiments, the channel width along the x-direction is approximately 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 8 mm, or 5 mm. In some embodiments, the channel length does not exceed approximately 10 mm or approximately 7 mm. In some embodiments, the channel width does not exceed approximately 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, or 5 mm.

[0163] In some embodiments, the channel width along the x-direction is 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 8 mm, or 5 mm. In some embodiments, the channel width does not exceed 10 mm or 7 mm. In some embodiments, the width of the channel does not exceed 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, or 5 mm.

[0164] In some embodiments, the distance between two adjacent channels or the distance from the edge of a channel to the edge of the flow pool device is about 0.5 mm to about 15 mm along the x-axis. In some embodiments, the distance between two adjacent channels or the distance from the edge of a channel to the edge of the flow pool device is about 1 mm to about 8 mm along the x-axis. In some embodiments, the distance between two adjacent channels or the distance from the edge of a channel to the edge of the flow pool device is about 2 mm to 6 mm along the x-axis.

[0165] In some embodiments, the distance between two adjacent channels or the distance from the edge of a channel to the edge of the flow pool device is 0.5 mm to 15 mm along the x-axis. In some embodiments, the distance between two adjacent channels or the distance from the edge of a channel to the edge of the flow pool device is 1 mm to 8 mm along the x-axis. In some embodiments, the distance between two adjacent channels or the distance from the edge of a channel to the edge of the flow pool device is 2 mm to 6 mm along the x-axis.

[0166] In some embodiments, the flow pool device may have more than one channel, and all channels may be the same size and shape. Figures 33A, 33E, and 33F illustrate embodiments of a flow cell apparatus having two channels of the same size and shape. In some embodiments, the flow cell apparatus may have channels of different sizes or shapes, or combinations thereof. Figures 31-32 illustrate embodiments of a flow cell apparatus having similar channel lengths but different channel widths.

[0167] In some embodiments, the channel may include a tapered portion that connects an open landing area to the body of the channel (e.g., Figures 2D, 5A, 30-32). The tapered region and its cone angle may be determined by the size of the open landing area to which it is connected and the width of the channel body. The size of the tapered region and its cone angle may be adjusted to facilitate efficient fluid transfer from the open landing area to the body of the channel. A tapered transition portion may connect the open landing area 241 to the body of the channel 250. A second tapered region may be used to connect the body of the channel 250 to the outlet 260. Figure 32 illustrates an embodiment of a flow pool device with a tapered transition portion 451a that connects the open landing area 441a to the body of the channel 452a. A second tapered region 453a may be used to connect the body of the channel 452a to the outlet 460a. Figure 38D illustrates the second tapered region 753a in different embodiments. The size and shape of the conical transition portion can vary depending on the various sequencing applications of the flow cell device, such as sample type, flow rate required during sequencing reaction, etc.

[0168] The size and shape of the conical transition portion 451a can vary depending on the application of the flow cell device. Figures 40A-40G show exemplary embodiments of connecting the body of the channel to a conical transition portion having an outlet of various sizes and dimensions. The sizes of the different components of the flow cell device are in millimeters. Figures 40A-40G are non-limiting examples of the flow cell device disclosed herein. Figure 40A is a side view of the flow cell device. Figure 40B shows a cross-sectional view along line A-A in Figure 40A. Figure 40C is a top view of the flow cell device. Figure 40D is a cross-sectional view along line B-B in Figure 40B. Figure 40E shows an extended view of region A in Figure 40B. Figure 40F shows an extended view of region C in Figure 40C. Figure 40G shows an extended view of region B in Figure 40D.

[0169] In some embodiments, the length of the tapered transition portion from the outlet to the channel body along the y-axis can be from about 3 mm to about 15 mm. In some embodiments, the length of the tapered transition portion from the outlet to the channel body along the y-axis can be from about 5 mm to about 12 mm. In some embodiments, the length of the tapered transition portion from the outlet to the channel body along the y-axis can be from about 6 mm to about 9 mm. In some embodiments, the length of the tapered transition portion from the outlet to the channel body along the y-axis can be from 3 mm to 15 mm. In some embodiments, the length of the tapered transition portion from the outlet to the channel body along the y-axis can be from 5 mm to 12 mm. In some embodiments, the length of the tapered transition portion from the outlet to the channel body along the y-axis can be from 6 mm to 9 mm.

[0170] In some embodiments, the cone angle, for example, the acute angle between the edge of the flow cell device and the edge of the conical region, is 25.1 degrees, as shown in FIG40B. In some embodiments, the cone angle may be in the range of about 15 degrees to about 40 degrees. In some embodiments, the cone angle may be in the range of about 20 degrees to about 30 degrees. In some embodiments, the cone angle may be in the range of 15 degrees to about 40 degrees. In some embodiments, the cone angle may be in the range of 20 degrees to about 30 degrees.

[0171] In some embodiments, each channel has its own corresponding open landing area or inlet or combination thereof, for example in FIGS. 32, 33A, 33F and 38A-38E. In some embodiments, two or more channels share a single open landing area or inlet or combination thereof, for example in FIGS. 33E and 41A-41C.

[0172] In some embodiments, the open landing area is directly connected to the body of the channel. In some embodiments, the open landing area is connected to the body of the channel in which there is no conical transition portion. FIGS. 38A-38E illustrate embodiments of the flow cell device disclosed herein. The flow pool device 112 includes a circular open landing area 741a that is directly connected to the body of a channel 752a without a tapered transition section. In this specific embodiment, the channel 752a begins where the open landing area ends, and the channel width is substantially the same as or exactly the same as the diameter of the open landing area. As shown in FIG39C, the size of the open landing area may differ from the embodiments in FIG38A-38E in one or more channels, such that the diameter of the open landing area is smaller than the width of the channel along the x-axis. When the diameter of the open landing area is small, there may be a tapered transition area 751a between the open landing area and the body of the channel.

[0173] The flow pool device 112 may include one or more inlets 240, 340a, 440a, 540a, 740a and one or more outlets 260, 460a, 560a, 760a and / or one or more cleaning outlets 270, 470a, 570a, 770a. Flow cell device 112 may include one or more channels 250, 350a, 450a, 550a, 750a. Channel 250 may extend from a corresponding inlet 240 to its corresponding outlet 260, thereby allowing fluid communication from inlet to outlet. Sequencing reagents may be introduced into flow cell device 112 through inlet 240, flow through channel 250 and interact with the sample located therein, and exit from outlet 260.

[0174] Flow cell device 112 may include one or more inlets 240, 340a, 440a, 540a, 740a and one or more outlets 560a. Channel 550a may extend from a corresponding inlet 540a to its corresponding outlet 560a, thereby allowing fluid communication from inlet 240 to outlet 260.Fluid communication to the outlet. Sequencing reagents can be introduced into the flow cell device 112 through inlet 540a, flow through channel 550a and interact with the sample located therein, and exit from outlet 560a.

[0175] In some embodiments, the flow cell device 112 further includes cleaning outlets 270, 470a, 570a, and 770a. The cleaning outlets can be in fluid communication with an open landing area to clean residues remaining thereon.

[0176] The cleaning outlets 270, 470a, 570a, and 770a can be located in one or more substrates. In some embodiments, the cleaning outlet 270 can be located as a side port (e.g., FIG. 2G) on a top substrate, bottom substrate, and / or intermediate substrate.

[0177] In some embodiments, the cleaning outlet includes a side port on one or more substrates. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-direction. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the y-direction. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the z-direction. In some embodiments, the side port extends at least along a direction inclined to the x-direction. In some embodiments, the side port extends at least along a direction inclined to the y-direction. In some embodiments, the side port extends at least along a direction inclined to the z-direction. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-y plane. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-z plane. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-y plane. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the y-z plane. In some embodiments, the side port extends at least along a direction inclined to the x-y plane. In some embodiments, the side port extends at least along a direction inclined to the x-z plane. In some embodiments, the side port extends at least along a direction inclined to the y-z plane.

[0178] The cleaning outlet 270 may be fluidly connected to the inlet 240. In some embodiments, the cleaning outlet is configured to be connected to a fluid drive device, such as a pump or vacuum device of a fluid control device, optionally via a connector, manifold, or both. The pump may be supplementary to the pump connected to the outlet 260. In some embodiments, the same fluid drive device, such as a pump, can be coupled to both the outlet and the clean outlet.

[0179] The distance from the clean outlets 270, 470a, 570a, 770a to the inlet 240 can be shorter than the distance to the outlet 260. The distance can be in the x-y plane. The shorter distance from the clean outlet to the inlet is designed to facilitate the transfer of liquid or gas from the open landing area to the clean outlet.

[0180] In some embodiments, the relative position of the cleaning exit 270 to the inlet or open landing area 241, 341a, 441a, 541a, 741a may differ. In some embodiments, the cleaning exit 270 may include a side port configured to mate with a connector or manifold device. In some embodiments, the side port extends at least along a direction perpendicular to or substantially perpendicular to the y-direction. In some embodiments, the side port extends at least along a direction perpendicular to or substantially perpendicular to the z-direction. In some embodiments, the side port extends at least along a direction inclined to the x-direction. In some embodiments, the side port extends at least along a direction inclined to the y-direction. In some embodiments, the side port extends at least along a direction inclined to the z-direction. In some embodiments, the side port extends at least along a direction perpendicular to or substantially perpendicular to the x-y plane. In some embodiments, the side port extends at least along a direction perpendicular to or substantially perpendicular to the x-z plane. In some embodiments, the side port extends at least along a direction perpendicular to or substantially perpendicular to the x-y plane. In some embodiments, the side port extends at least along a direction perpendicular to or substantially perpendicular to the y-z plane. In some embodiments, the side port extends at least along a direction inclined to the x-y plane. In some embodiments, the side ports extend at least along a direction inclined to the x-z plane. In some embodiments, the side ports extend at least along a direction inclined to the y-z plane.

[0181] In such embodiments, the cleaning outlets 270, 470a, 570a, and 770a are not directly connected to the corresponding open landing areas, but are connected via tapered transition portions therebetween, such as in FIG. 2D.

[0182] The distance from the cleaning outlet (from the proximal end of the cleaning outlet) to the nearest edge or center of the open landing area can be 0 mm or about 10 mm. When the cleaning outlet is not directly below the open landing area, the distance from the cleaning outlet to the nearest edge or center of the open landing area can be about 0 mm to about 20 mm. When the cleaning outlet is not directly below the open landing area, the distance from the cleaning outlet to the nearest edge or center of the open landing area can be about 0 mm to about 15 mm. When the clean exit is not directly below the open landing area, the distance from the clean exit to the nearest edge or center of the open landing area can be from about 3 mm to about 10 mm.

[0183] In some embodiments, reagent residues may be present, such as menisci remaining on the walls of the orifice of the open landing area or inlet, or combinations thereof. If such residues are not removed, unintentional mixing may occur when subsequent reagents are delivered to the open landing area, and thus contaminate the sequencing reaction in the channel. Liquid washing alone may not be effective in removing residual reagents such as menisci, and therefore multiple rinses with washing solution may be required to completely remove them from existing flow cell systems.The residues increase washing time and washing costs. A fluidly connected cleaning outlet 270 can advantageously facilitate the time- and cost-effective removal of such residues. In some embodiments, mechanical driving force can be applied through the cleaning outlet, for example by a pump or inlet vacuum device, to completely remove such residual reagents from the open landing area. Therefore, the time and washing volume required to remove residues to achieve a satisfactory level of contamination can be effectively improved from existing flow cell devices.

[0184] The size and shape of the cleaning outlet can be customized to suit different sequencing applications. Although the cleaning outlet is shown as a cylindrical shape in Figures 2D-2F, it can be made into different shapes, such as cones, inverted cones, etc. In some embodiments, the size and shape of the cleaning outlet can be the same as the size and shape of the outlet. In some embodiments, the size of the cleaning outlet can differ from the size of the outlet by no more than about 10%, 20%, or 30%. In some embodiments, the diameter of the cleaning outlet in the x-y plane is about 0.3 mm to about 10 mm. In some embodiments, the height of the cleaning outlet in the z-direction is the same as the height of the bottom substrate. In some embodiments, the height of the cleaning outlet is about 0.3 mm to about 3 mm. In some embodiments, the height of the cleaning outlet is from about 0.5 mm to about 1 mm. In some embodiments, the diameter of the cleaning outlet in the x-y plane is from 0.3 mm to 10 mm. In some embodiments, the height of the cleaning outlet in the z-direction is the same as the height of the bottom substrate. In some embodiments, the height of the cleaning outlet is from 0.3 mm to 3 mm. In some embodiments, the height of the cleaning outlet is from 0.5 mm to 1 mm.

[0185] The size and shape of the inlet and outlet can be customized to suit various sequencing applications. For example, the size and shape can be determined based on specific sequencing applications, such as minimum flushing volume, contamination threshold, flow cell parameters (e.g., width of flow cell channels) or dispenser parameters (e.g., size of dispensing tip). As a non-limiting example, the inlet can be cylindrical with walls extending along the z-direction and orthogonal to the substrate, for example, as shown in FIG33C. In some embodiments, the inlet 240 can be connected to the cleaning outlet 270 at the bottom of the cylindrical void / hole. In some embodiments, the inlet 440a can be connected to the cleaning outlets 470a, 570a at the bottom of the cylindrical void / hole. In different embodiments, the shape of the inlet can be different. For example, the inlet can have an inverted cone shape, with a wider opening at the top that narrows towards the channel to reduce reagent residue that may remain in the inlet. Figure 33D illustrates an embodiment with an inlet in a cylindrical shape. In embodiments without a cleaning outlet, the flow cell device does not include a connection extending from the inlet to the cleaning outlet. In another embodiment,As shown in Figure 33E, inlet 540a may be part or all of the open landing area. In yet another embodiment of the flow cell device, as shown in Figure 33F, the inlet may be a recess of various sizes or shapes on the intermediate substrate or on the intermediate and bottom substrates, the inlet being fluidly connected to the channel.

[0186] In another embodiment, as shown in Figure 33E, inlet 540a may include an open landing area or a portion thereof, but not other structural elements of the flow cell device. Figures 41A-41C show the embodiment of Figure 33E from different views. Figure 41A is a top view of the flow cell device. Figure 41B shows three different substrates in perspective, and Figure 41C shows the bottom substrate, intermediate substrate, and top substrate. In yet another embodiment of the flow cell device, as shown in Figure 33F, the inlet may be a recess of various sizes or shapes on the intermediate substrate or on the intermediate and bottom substrates, the inlet being fluidly connected to the channel. Specification 28 / 105 pages 51 CN 121057626 A

[0187] The diameter of the inlet, for example, the widest dimension in the x-y plane, can be in the range of about 3 mm to about 11 mm. As another example, the height of the inlet along the z direction can be the total height of the top substrate and the intermediate substrate, and it can be in the range of about 1 mm to 12 mm.

[0188] The diameter of the outlet or cleaning outlet in the x-y plane can be in the range of about 0.3 mm to about 4 mm. In some embodiments, the diameter of the outlet can be in the range of about 0.4 mm to about 2 mm, and the outlet can be cylindrical in shape.

[0189] The diameter of the inlet, for example, the widest dimension in the x-y plane, can be in the range of 3 mm to 11 mm. As another example, the height of the inlet along the z direction can be the total height of the top substrate and the intermediate substrate, and it can be in the range of 1 mm to 12 mm.

[0190] The diameter of the outlet or cleaning outlet in the x-y plane can be in the range of 0.3 mm to 4 mm. In some embodiments, the diameter of the outlet can be in the range of 0.4 mm to 2 mm, and the outlet can be cylindrical in shape.

[0191] The size and shape of the inlet and outlet can be customized to suit various sequencing applications. For example, the size and shape can be determined based on specific sequencing applications, such as minimum flushing volume, contamination threshold, flow cell parameters (e.g., width of flow cell channels) or dispenser parameters (e.g., size of dispenser tips).

[0192] Figures 30-32 and 33A-33F illustrate flow cell devices having two to three substrates forming one or two channels, and each channel having a corresponding inlet and outlet. However, in different embodiments, the number of substrates, channels, inlets, and outlets can vary. In some embodiments, the number of substrates, channels, inlets, and outlets can be greater than 0.Any integer. In some embodiments, the flow cell device herein has 2, 4, 6, 8, 10, or even more channels.

[0193] In some embodiments, the flow cell device may include one or more seals 290 that help prevent leakage between structural elements of the flow cell device or between the flow cell device and other devices (e.g., manifolds, connectors, pumps, etc.) to which the flow cell device is connected. Leakage may damage or contaminate the sequencing system or the sample attached to the flow cell device. For example, without one or more seals, leakage may occur between two adjacent layers in the top or bottom substrate. As another example, leakage may occur between the flow cell device and its manifold connection, or between the flow cell device and the connector connecting the flow cell device and the manifold.

[0194] The one or more seals may comprise one or more mechanical seals. The one or more seals may comprise: a gasket 267, a manifold or connector, a portion of a manifold device, or a combination thereof. The one or more seals may comprise one or more gaskets. The one or more seals may comprise a flexible material, such as rubber, that deforms under pressure that meets a predetermined threshold.

[0195] In some embodiments, the one or more seals may be positioned along the y-axis at one end of the substrate, as shown in Figures 2A-2C.

[0196] In some embodiments, the flow pool device may include increasing the thickness of the substrate (e.g., Figures 2A-2C) at the second portions 226 and 236, thereby advantageously facilitating attachment of the one or more seals to the substrate and thus enabling improved sealing of fluid communication compared to a flow pool device without increasing the thickness of the substrate. In some embodiments, the increased substrate thickness, combined with fluid channels having a z-offset in the two portions, also provides clearance for the fluid dispensing element, for example, dispensing the tip into an open landing area. In other words, the flow pool device herein (e.g., having a variable thickness in the top substrate) may provide clearance above the top surface of the top substrate and avoid blocking the movement of the dispensing tip at the same z level along the x-y plane.

[0197] In some embodiments, the one or more seals include a first seal whose thickness along the z direction is comparable to the thickness of the second portion 226, for example, the thickness of the top substrate in Figure 2A. The one or more seals may, as described on page 29 / 105 of CN 121057626 A, include a second seal, the thickness of which along the z-direction is comparable to the thickness of the bottom substrate in the second portion 236. In some embodiments, the thickness of the second seal along the z-direction is greater than the thickness of the bottom substrate in the first portion. The one or more seals may include various widths along the y-axis and / or x-axis.

[0198] As shown in FIG3A, the flow cell device 112 may further include a frame 295 that covers at least a portion of the one or more substrates. In some embodiments, the frame 295 is mechanically secured to the one or more seals. The frame may comprise plastic, metal, polymer, glass, or a combination thereof. The frame may be configured to facilitate positioning of the one or more substrates relative to a connector or manifold in fluid communication with the flow cell device. In some embodiments, the frame may cover a portion of the top substrate and / or bottom substrate, as shown in FIG3A. In some embodiments, the frame may keep the top substrate and / or bottom substrate exposed for imaging and heat transfer purposes, as shown in FIG3B. A larger gasket, which is part of instrument 242; an intermediate gasket, which is part of flow cell consumable 244; and a plastic frame, which is part of flow cell consumable 295, are also shown in FIG3B.

[0199] In some embodiments, the flow cell system may further include a manifold or connector 299 that interfaces with the flow cell device 112. The manifold or connector may include one or more fluid passages 298. The one or more fluid passages 298 may be in direct or indirect fluid communication with the one or more channels 250. In some embodiments, a manifold or connector includes one or more fluid passages 298 that are in fluid communication with one or more open landing regions 241, 341a, 441a, 541a, 741a. For example, in Figures 4A-4D, the manifold or connector includes a corresponding fluid passage leading to a region that, when connected to a flow cell device, becomes a complete circular open landing region 241. In other words, a portion of the entire open landing region 241, 341a, 441a, 541a, 741a may be contained within a manifold or connector 299, while another portion of the entire open landing region 241 may be contained within a substrate. As shown in Figures 4A-4D, partially or completely removing the open landing region from the flow cell device can advantageously increase the length of the one or more channels along the y-axis, thus increasing the imaging area compared to a flow cell device with an open landing region that cannot be used as an imaging area. Partial or complete movement to the open landing area of ​​the manifold or connector can be combined with other embodiments herein to increase the imaging area in a variety of sequencing and imaging applications.

[0200] In some embodiments, the manifold or connector is configured to be in sealed fluid communication with the one or more channels when a force or pressure satisfying a predetermined threshold is applied thereto. The force or pressure may be at least along the y-axis. In some embodiments, the pressure applied to structural elements of the flow cell system, such as gaskets or manifolds, may be 0 kPa to 500 kPa, 0 kPa to 280 kPa, 0 kPa to 250 kPa, or 0 kPa.The force applied to the structural elements of the flow cell system, such as gaskets or manifolds, may be in the range of 0 N to 80 N, 0 N to 60 N, 2 N to 50 N, or 5 N to 30 N. In some embodiments, some or all of the structural elements of the flow cell system may be in a vacuum configuration, thus applying a pressure or force that satisfies a threshold for sealing the fluid communication between the flow cell device and the manifold. In some embodiments, the pressure threshold is in the range of 100 kPa to 500 kPa. In some embodiments, the pressure threshold is in the range of 150 kPa to 300 kPa. In some embodiments, the force threshold is in the range of 0.1 N to 35 N. In some embodiments, the force threshold is in the range of 1 N to 25 N.

[0201] The one or more fluid passages 298 may extend along the y-axis, where pressure is applied along the y-axis, for example, FIG. 4A. The one or more fluid passages extend along the x-axis, where pressure is applied along the x-axis, for example, FIG. 5A. One or more reference features 297 and manifold interface 271 are also shown in FIG. 5A. In some embodiments, the one or more fluid passages may extend in any direction in the x-y plane, y-z plane (FIG. 12A), or three dimensions. FIG. 5B, 5C, and 5D are also shown. In FIG. 5B, a centering alignment pin 272 and a reference established by centering alignment feature 273 are also shown. A hole in slot alignment feature 274 is also shown in FIG. 5C. A pin hole alignment feature 275 is also shown in FIG. 5D.

[0202] In some embodiments, a single fluid passage may correspond to and be fluidly connected to only the corresponding channel to minimize contamination across channels. In some embodiments, a single fluid passage may be fluidly connected to multiple channels. Advantageously, the flow cell device described herein can be connected to different connectors or manifolds with different configurations (sealed fluid communication) to optimize the flexibility of using the flow cell device for different sequencing applications or chemical protocols. For example, as shown in FIG. 7B, different channels of the flow cell device can be fluidly connected to the same kit via a manifold or connector 299. Alternatively, as shown in Figure 6B, different channels of the flow cell device can be in fluid communication with different reagent kits using different manifolds or connectors 299.

[0203] In some embodiments, the one or more seals interface with a manifold, connector, or fluid control device to allow sealed fluid communication with the one or more channels, as shown in Figures 3A-3B, 5A, and 6A-6C. Such interfaces can be direct or indirect. In the case of direct interfaces, the one or more seals are directly connected to the manifold or flow control device. In the case of indirect interfaces, there may be connectors or fittings in between.

[0204] In some embodiments, the one or more seals may be used as part of a connector or manifold. In some embodiments, the one or more seals may include: a connector, a manifold, a portion of a manifold, or a combination thereof.

[0205] In some embodiments, the one or more seals include a protective sleeve seal 290 that covers at least a portion of the flow cell device 112 in the x-y plane. In some embodiments, the protective sleeve seal also covers one end of the flow cell device in the x-z plane, for example, in FIG. 9A.

[0206] In some embodiments, the one or more seals include a membrane seal 290. In some embodiments, the membrane seal or sleeve may overlap two surfaces of the flow cell device with a manifold or connector at the top and / or bottom of the flow cell device. The membrane seal may be flat or conform to the flatness of the surfaces to which it overlaps. FIG. 9B-9E show embodiments of a flow cell device with a membrane seal 290. Compared to other sealing geometries (e.g., O-rings at the ends of a substrate), membrane seals may advantageously require less sealing force or pressure. In some embodiments, the membrane seal can cover at least a portion of the manifold and a portion of the substrate along a path length along the y-axis, for example, the path length along the y-axis can be several millimeters or longer. The membrane seal can also include a thin cross-section along the z-axis and / or x-axis, thereby having relatively high flow resistance. The effect of air infiltrating from around the seal on the sealed fluid communication is negligible. Another advantage of the membrane seal is that the internal negative pressure acts over a larger area, and this force multiplication over a large area can more effectively overcome any stiffness in the loose sleeve and maintain the thin cross-section in vacuum applications. The membrane seal can reduce or eliminate flow between individual fluid channels in a flow cell device, thereby effectively sealing two channels independently. As shown in Figure 9E, gasket material can be included at the finger-cut location to separate nearby channels and enable a seal between them. The finger-cut area separating the fluid path 282 is also shown in Figure 9B. A plastic gasket manifold 285 and a polymer gasket sleeve 284 per lane are also shown in Figure 9E.

[0207] The materials of the membrane seals or other seals disclosed herein may be compliant, such that the sealing force increases with increasing vacuum, for example, proportionally. The path length along the y-axis may be of various lengths from 1 mm to 4 cm. The path length along the y-axis may be of various lengths from 2 mm to 2 cm. The path length along the y-axis may be of various lengths from 2 mm to 1 cm. As shown in FIG9A, the path length may cover all widths of the flow cell device along the x-axis. FIG9D shows an extended view of the interface between the manifold or connector 299 and the flow cell device 112, which interfaces with the membrane seal 290.Figure 9D. In some embodiments, the one or more seals may additionally include an adhesive or shrink sealing element to enhance the seal. Path length 283 is also shown in Figure 9D.

[0208] In some embodiments, the one or more seals comprise an L-shaped seal extending along the z-axis and y-axis. As shown in Figure 8, the L-shaped seal extends along the y-axis and into a corresponding channel in one or more channels. Pressure or force may be applied along the y-axis to the L-shaped seal to allow for sealed fluid communication between the flow pool device and the manifold. The one or more seals may be configured to interface with a manifold or connector, thereby allowing sealed fluid communication between the flow pool device and the manifold. In some embodiments, the seal extending along the z-axis and y-axis may be of various shapes similar to an L-shape, such as a C-shape. The size of the arms of the L-shape may also be varied, for example, covering at least a portion or all of the thickness of the substrate. A flexible baffle inserted into the flow pool channel 281 is also shown in Figure 8.

[0209] In some embodiments, the one or more seals include a diagonal washer having a fluid passage extending in the y-z plane. FIG12A shows a side view of an embodiment of a flow pool device and a diagonal washer 288 connectable to the flow pool device. The diagonal washer may interface with an end of a top substrate and a bottom inner surface 252 of a bottom substrate, as shown in FIG12A. Alternatively, the diagonal washer may interface with an end of a bottom substrate and a top inner surface 251 of a top substrate. The washer may have a thickness along the z-axis that does not impede movement of the dispensing tip toward an open landing area, for example, in the x-y plane. The acute angle between the channel and the fluid passage may vary from 0 degrees to 85 degrees. The acute angle between the channel and the fluid passage may vary from 10 degrees to 65 degrees.

[0210] FIG12B shows a top view of an embodiment of a flow pool device having a manifold or connector 299 that is structurally and functionally similar to the diagonal washer in FIG12A. Figure 12C shows a side view of the flow cell assembly in Figure 12B. Figure 12D is an extended view of a manifold or connector 299 that interfaces with the channel 250 of the flow cell assembly in Figure 12C. Figure 12E shows a manifold or connector not connected to the flow cell assembly. The manifold or connector may interface with the end of the bottom substrate and the top inner surface 251 of the top substrate, as shown in Figure 12C. As shown in Figure 12C, connecting the diagonal washer, manifold, or connector to the flow cell assembly can advantageously provide a clearance above the top surface 261 of the top substrate, thereby facilitating easy and efficient movement of the dispensing tool to the inlet or open landing area of ​​the flow cell assembly. Alternatively, the manifold or connector may be connected to...The ends of the top substrate and the bottom inner surface 252 of the bottom substrate intersect. When a force or pressure comprising a y-axis component satisfying a first threshold and a z-axis component satisfying a second threshold is applied, the diagonal washer, manifold, or connector 299 may allow sealed fluid communication from the fluid passage 298 to the one or more channels 250. In some embodiments, the diagonal washer, manifold, or connector may include a handle for applying force or pressure on the diagonal washer, as shown in Figures 12C and 6C. The force or pressure may be applied along the direction in which the handle extends. The force or pressure may be applied in different 3-dimensional directions. The force or pressure may be at least along the y and z directions. An optional self-alignment feature 277 is also shown in Figure 6C.

[0211] In some embodiments, the manifold or connector 299 may include a connector core 299_1 and a washer overmolding 299_2 on at least a portion of the connector core. For example, the overmolding 299_2 may cover the interfacing area of ​​the connector 299 with the substrate of the flow cell device 112, as shown in Figure 12D. In some embodiments, the manifold or connector may include a connector core 299_1 and a separate gasket, for example, in an "L" shape or various other shapes, which may be assembled with the connector core 299_1.

[0212] In some embodiments, the top substrate and bottom substrate of the flow cell device may be laterally offset from each other along the y-axis, and the manifold or connector 299 may be positioned on top of the bottom substrate, as shown in Figures 12F-12H. In such embodiments, the manifold or connector may be fixedly attached to the one or more substrates. For example, the manifold or connector 299 may be laminated to the substrate using pressure-sensitive adhesive or various bonding or adhesive methods. The fluid passage 298 in the manifold or connector may be in direct or indirect sealed fluid communication with one or more channels of the flow cell device. As shown in Figure 12G, the fluid passage 298 in the manifold or connector is in direct fluid communication with an open landing area and a channel without a clean outlet 270. The fluid passage 298 may include an end connected to a fluid control device, such as a vacuum device. As shown in Figure 12G, this end of the fluid passage may be in a plane orthogonal to the x-z plane. Alternatively, the fluid passage may extend from the one or more channels and exit the manifold or connector from one side in a plane orthogonal to the y-z plane (not shown). Alternatively, the fluid passage may extend from the one or more channels and exit the manifold or connector from the top or bottom in a plane orthogonal to the x-y plane (not shown). An intermediary layer 289 and an optional adhesive or seal 292 are also shown in Figure 12G.

[0213] In some embodiments, the flow pool system herein may include an intermediary layer defining the oneOne or more channels and open landing areas. As shown in FIG12G, an intermediary layer may be located between the manifold or connector and the bottom substrate. In some embodiments, when the manifold or connector is positioned below the top substrate, the intermediary layer may be located between the manifold or connector and the top substrate.

[0214] In some embodiments, at least some portions of the manifold may comprise plastic.

[0215] In some embodiments, adhesive seals may be used in some interface areas between the manifold or connector and the substrate. In some embodiments, adhesive seals may be applied depending on the material and surface properties of the substrate and the manifold or connector. For example, as shown in FIG12G, optional adhesives or seals may be added when the manifold or connector has an inclined surface facing the end of the top substrate.

[0216] In some embodiments, open landing areas 241, 341a, 441a, 541a, 741a may be contained in the manifold or connector, rather than in the one or more substrates, as shown in FIG12F-12G.

[0217] In some embodiments, two similarly sized substrates, a top substrate and a bottom substrate, are laterally offset along the y-axis to create two engagement regions for the manifold or connector. As shown in FIG12G, one engagement region is located near the open landing region. As shown in FIG12F, the other engagement region is located at the other end of the flow cell device near the outlet and is configured to engage the lower manifold or connector 299. Alternatively, as shown in FIG12H, a larger bottom substrate can be used, so that both engagement regions are configured to securely attach the manifold or connector to the top of the bottom substrate, rather than the lower manifold or connector. In some embodiments, the thickness of the manifold along the z-axis can be maintained comparable to the thickness of the top substrate, thereby providing clearance for distributing the tip to the open landing region.

[0218] FIG13 illustrates the thermoplastic connector 294 and the thermoplastic seal 296.

[0219] In some embodiments, the length of the bottom substrate along the y-axis can range from 50 mm to 120 mm. In some embodiments, the length of the bottom substrate along the y-axis can range from 60 mm to 110 mm. In some embodiments, the length of the bottom substrate along the y-axis can be in the range of 75 mm to 100 mm. In some embodiments, the length of the bottom substrate along the y-axis can be in the range of 85 mm to 100 mm.

[0220] In some embodiments, the one or more seals comprise a thermoplastic connector and a thermoplastic seal mounted on the thermoplastic connector. The thermoplastic seal can deform under pressure changes, temperature changes, or both. The thermoplastic seal can comprise one or more materials different from the thermoplastic connector. When a force or pressure satisfying a predetermined threshold is applied, the thermoplastic seal can achieve sealed fluid communication between the channels and manifolds of the flow pool device. The force or pressure can include at least a y-axis component.

[0221] It is worth noting that the threshold force or pressure used to seal the fluid communication may be different or the same in different embodiments of the flow cell device. It is also worth noting that various mechanisms can be used to maintain a constant application of force or pressure over a predetermined time period. The predetermined time period can range from less than one second to several hours. In some embodiments, the predetermined time period includes a time window during which reagents communicate between the flow cell device and other structural elements of the flow cell system, such as manifolds or connectors. In some embodiments, the predetermined time period includes a time window during a sequencing run.

[0222] The force or pressure may be at least along the y-axis. In some embodiments, the pressure applied to seal the fluid communication between the flow cell device and other elements, such as gaskets or manifolds, may be in the range of 0 kPa to 500 kPa, 0 kPa to 280 kPa, 0 kPa to 250 kPa, or 0 kPa to 220 kPa. In some embodiments, the pressure applied to seal the fluid communication between the flow cell device and its components, such as gaskets or manifolds, may not exceed 100 kPa, 150 kPa, 180 kPa, 200 kPa, 300 kPa, or 400 kPa. In some embodiments, the force applied to the flow cell system, such as gaskets or manifolds, may be in the range of 0 N to 80 N, 0 N to 60 N, 2 N to 50 N, or 5 N to 30 N. In some embodiments, the force applied to the flow cell system, such as gaskets or manifolds, may not exceed 20 N, 25 N, 20 N, 35 N, 40 N, 45 N, 50 N, 55 N, 60 N, 70 N, 80 N, 100 N, or 200 N. In some embodiments, some or all of the structural components of the flow cell system may be in a vacuum configuration, thus applying a pressure or force that satisfies a threshold for sealing the fluid communication between the flow cell device and the manifold. In some embodiments, the pressure threshold is in the range of 50 kPa to 500 kPa. In some embodiments, the pressure threshold is in the range of 150 kPa to 300 kPa. In some embodiments, the force threshold is in the range of 0.1 N to 35 N. In some embodiments, the force threshold is in the range of 1 N to 25 N.

[0223] In some embodiments, the one or more seals may include a first connector having a top portion that is slidable on a top surface 261 of a top substrate. The one or more seals include a second connector having a bottom portion that is slidable on a bottom surface 262 of a bottom substrate. The top portion may be connected to a first side portion of the first connector, the first side portion being configured to be in x-The flow cell device is interfaced with the end of the flow cell device in the z-plane. The bottom portion may be connected to a second side portion of the second connector, which is configured to interface with the end of the flow cell device in the x-z plane. The top or bottom portion is connected to its corresponding side portion to form an integrated connector. Pressure or force satisfying a predetermined threshold on the first and second side portions may be configured to allow the first and second connectors to slide relative to the flow cell device under deformation, thereby enabling the one or more channels to be in sealed communication with the fluid passage defined between the top and bottom connectors.

[0224] Figures 14A-14D show non-limiting examples of a first connector 301 and a second connector 305, the first connector having a top portion that can slide on the top surface of a top substrate, and the second connector having a bottom portion that can slide on the bottom surface of a bottom substrate. The specific geometry of the first and second connectors may vary in different applications. The sliding surfaces of the top and bottom portions may be generally flat to allow smooth sliding relative to the flow cell device. In this particular embodiment, the flow cell device includes a port located at the bottom surface of the flow cell to allow fluid communication between the fluid passage and the one or more channels. In some embodiments, the port may open at the top surface, at one end of the flow cell device along the y-axis (or in the x-z plane), or at either side of the flow cell device along the x-axis (or in the y-z plane).

[0225] The first connector may include a smooth top surface as shown in Figures 14C-14D to provide clearance for the dispensing tip to travel to the open landing area. Figures 14B and 14C show the connector in disconnected and connected positions with the flow cell device. A force application mechanism may be used to actuate the connector to enable accurate connection or disconnection with the sealed fluid communication. The force application mechanism may include a motor. The force application mechanism may be controlled by the sequencing system, for example, by applying force or pressure via the motor over a predetermined time period, such as during a sequencing run, using software programs executable on the computer processor of the sequencing system. A pipette 302 and a pipette travel area 303 are also shown in Figure 14C. A bottom port on the flow cell is also shown in Figure 304.

[0226] In some embodiments, the one or more seals comprise a semi-rigid or deformable material that deforms under pressure or force. In some embodiments, a semi-rigid or deformable material is configured to recover its shape before deformation when pressure or force is removed.

[0227] In some embodiments, the top substrate or the bottom substrate includes one or more inclined ends. In some embodiments, the tips of the inclined ends may press against the one or more seals, for example, FIG. 10. The inclined ends can facilitate fluid communication through a seal that requires less pressure or force. The ramp may be in the y-z plane. Pressing against one or more sealsPage 34 / 105, CN 121057626 A. The tips on the multiple seals may be on the top or bottom surface of the top or bottom substrate, for example, 251, 252, 261, 262. A semi-sharp corner of the flow pool pressed into the gasket 286 is also shown in Figure 10.

[0228] In some embodiments, each of the inclined ends may interface with an inclined manifold or connector, such as in Figure 11. The one or more inclined ends may include a first acute ramp angle relative to the y-axis, and wherein the inclined manifold or connector includes a second acute ramp angle relative to the y-axis. The first acute ramp angle may be different from the second acute ramp angle. The first acute ramp angle may be substantially the same as the second acute ramp angle. In this case, the inclined manifold or connector includes a ramp complementary to the inclined end of the flow pool device. A rigid flared manifold 287 is also shown in Figure 11.

[0229] In some embodiments, the flow cell device 112 further includes one or more reference features configured to position the flow cell device relative to a manifold or connector, sample stage, or sequencing system. The one or more reference features may include at least one alignment feature positioned at or near a center point along the x-axis. The one or more reference features include at least one alignment feature positioned at or near an end of the one or more substrates along the y-axis. As shown in Figures 5A-5D, the one or more reference features 297 include cavities extending through the one or more substrates and connectable to pins or supports. In some embodiments, the one or more reference features include recesses (e.g., Figure 5A) extending through the one or more substrates, which are connectable to pins or supports. In some embodiments, the one or more reference features may include various features that can be coupled together for alignment purposes, including but not limited to recesses, clips, side arms, etc.

[0230] In some embodiments, the cleaning outlet may extend at least in the x-y plane. For example, the cleaning outlet 270 extends in the x-y plane as a side port, as shown, for example, in Figures 2D, 4, and 5A. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the y-direction. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the z-direction. In some embodiments, the side port extends at least along a direction inclined to the x-direction. In some embodiments, the side port extends at least along a direction inclined to the y-direction. In some embodiments, the side port extends at least along a direction inclined to the z-direction. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-y plane. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-z plane. In some embodiments...In embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the x-y plane. In some embodiments, the side port extends at least along a direction perpendicular to or nearly perpendicular to the y-z plane. In some embodiments, the side port extends at least along a direction inclined to the x-y plane. In some embodiments, the side port extends at least along a direction inclined to the x-z plane. In some embodiments, the side port extends at least along a direction inclined to the y-z plane.

[0231] Such orientation of the cleaning outlet can advantageously be compatible with different flow cell shapes, sizes, and / or channels of different shapes or sizes. For example, Figure 5D illustrates how to align different flow cell devices with the same sample stage using the reference features disclosed herein, and how to extend a flow cell device with two channels in the x-y plane, especially along the x-axis, without changing the existing cleaning outlet or adding additional cleaning outlets for other channels or wider channels. The inclined portion of a single channel can also be shaped to allow the use of a shared cleaning outlet between two or more channels. In some embodiments, the positioning and orientation of the clean outlet, for example as shown in Figures 2D, 4, and 5A, advantageously allows different flow cell devices to be sequenced without requiring changes to or additions to the fluid connections of the manifold / connector and / or fluid control device, thereby enabling convenient and efficient switching between different sequencing applications. Additionally, the positioning and orientation of the clean outlet, for example as shown in Figures 2D, 4, and 5A, advantageously allows for convenient and efficient scaling of the flow cell device to increase the number of channels and samples to be sequenced, thereby allowing improved sequencing throughput using the same sequencing system. Figure 2E shows a side view of the flow cell device in Figure 2D, and Figure 2E also shows a cross-sectional view of the flow cell device in Figure 2D at AA. Figure 2G shows a detailed view of Figure 35 / 105, page 58, CN 121057626 A 2F.

[0232] In some embodiments, the manifold or connector 299 includes a top portion or a bottom portion that extends along the z-axis beyond the plurality of substrates and covers at least a portion of one or more substrates in the x-y plane. The top or bottom portion of the manifold may be located at a first portion, a second portion, or both of the one or more channels 250. Figures 6A-6C show a non-limiting example of a flow pool device 112 having a manifold or connector 299. The manifold may include a connector 299' that connects the flow pool device 112 and other elements of the manifold or connector 299. Alternatively, the connector 299' may be a structurally separate component that functions to connect the flow pool device and other elements of the manifold.

[0233] The connector 299' may include a top portion and a bottom portion that define an opening therebetween. The opening may be connected to the channel250 Direct sealed fluid communication. In some embodiments, each channel may be in fluid communication with the fluid passage 298 of the manifold in a connected position, as shown in FIG6B. FIG6C shows the connector 299' in a position separated from the flow cell device 112. The flow cell device may include an optional gasket at the interface between the flow cell device and the manifold / connector 299.

[0234] The flow cell system may include a force application mechanism, including but not limited to a motor, an electromagnetic actuator, a spring, a linkage, or a combination thereof. In some embodiments, the manifold or connector 299 may be driven by the force application mechanism to connect to or disconnect from the flow cell device. The force application mechanism may be controlled by the sequencing system to enable connection or disconnection with the flow cell device, thereby achieving sealed fluid communication between the flow cell device and the fluid control device. In some embodiments, the manifold or connector 299 may be part of the manifold device or fluid control device of the flow cell system.

[0235] In some embodiments, the flow cell system further includes one or more tubes that interface with the manifold or connector 299 and the flow cell device 112. A tube may be positioned therebetween. Each of the one or more tubes 291 may include a wall surrounding a lumen 551a. The lumen 551a may be in fluid communication with the one or more channels 250 of the flow cell device and the one or more fluid passages 298 of the manifold or connector 299. An embedded tube may advantageously provide an improved seal compared to a seal at the end face of the substrate. An optional O-ring may be included to further improve the seal between the embedded tube and the flow cell device. Figures 7A-7D show non-limiting examples of flow cell devices with embedded tubes. In the embodiments shown in Figures 7B-7D, the flow cell device 112 may be integrated with the manifold or connector 299 such that they are fixedly attached to each other with sealed fluid communication therebetween to facilitate easy operation of the flow cell device and easy and leak-proof connection to fluid control devices. For example, the integrated flow cell and connector may interface with an instrument-side connector. Such interfaces may include interfaces that are easy to disconnect, for example, as shown in Figures 6B-6C. An optional O-ring 278 and a tube embedded in the flow cell 279 are also shown in Figure 7D.

[0236] In some embodiments, the applied pressure or force may be customized according to the different size, shape, material, or other characteristics of the flow cell system. In some embodiments, the pressure applied to the structural elements of the flow cell system, such as gaskets or manifolds, may be in the range of 0 kPa to 320 kPa, 0 kPa to 280 kPa, 0 kPa to 250 kPa, or 0 kPa to 220 kPa. In some embodiments, some or all of the structural elements of the flow cell system may be in a vacuum configuration.Therefore, a pressure is applied that satisfies a threshold for sealing the fluid communication between the flow cell device and the manifold. In some embodiments, the pressure threshold is in the range of 150 kPa to 300 kPa. In some embodiments, the force threshold is in the range of 0.1 N to 35 N. In some embodiments, the force threshold is in the range of 1 N to 25 N.

[0237] In some embodiments, the force applied to the structural elements of the flow cell system, such as gaskets or manifolds, can be in the range of 0 N to 50 N, 0 N to 40 N, 5 N to 30 N, 5 N to 25 N, 1 N to 25 N, or 5 N to 15 N. In some embodiments, some or all of the structural elements of the flow cell system can be in a vacuum configuration, thus applying a force that satisfies a threshold for sealing the fluid communication between the flow cell device and the manifold. Specification 36 / 105 pages 59 CN 121057626 A

[0238] It is worth noting that the different embodiments of the flow cell device and the features disclosed corresponding to such embodiments are not limited to the corresponding embodiments disclosed therein. Instead, embodiments and their corresponding features can be combined together for various customized needs. As a non-limiting example, FIG12H includes reference feature 297 as shown in the embodiments of FIG5A-5D. As another example, the one or more substrates in the embodiments shown in FIG12A-12H may include a bottom or top substrate having one or more layers 221, 231 as shown in FIG2A-2C. To date, in one example, the one or more substrates in FIG12A-12H may include an inclined end as shown in FIG11. To date, in another example, as shown in FIG2D, 4A-4B and 12F, the open landing area may be entirely on the flow cell device, partially on the flow cell device, or entirely on the manifold or connector. As shown in FIG4B, the connector includes a coupled end cap 255 that can interface with the flow cell device, and the open landing area may be separated between the end cap and the flow cell device. FIG4B also shows a pipette landing pad separated between the end cap and the flow cell 256, and a coupling interface 257. FIG4C shows the connectorized end cap 255 in the disconnected position, and the presence of a face seal gasket 290. Figure 4D shows a pipette landing pad fully located on end cap 263, and a coupled or connectorized interface 293.

[0239] As shown in Figure 33C, one or more inner surfaces of inner surface 521a may be coated with a first coating 522a.

[0240] In some embodiments, the channel is configured to allow fluid, such as a liquid reagent, to flow through an air gap between the fluid and the channel. In some embodiments, the air gap may contain a gas mass. The air gap may be similar to a liquid reagent, for example, entering the channel through an inlet and then exiting from an outlet and / or a clean outlet. Alternatively, the air gap may be from a device such as a flow cell.Other openings, such as the outlet or clean outlet, are introduced. The air gap can be mechanically driven by one or more structural elements of the fluid control device described herein. As an example, the air gap can be drawn into the channel through the inlet by a mechanical force applied at the outlet, such as by a pump or vacuum device. As another example, the air gap can be purged through the inlet by a pump or the like.

[0241] The volume of the air gap can vary depending on the geometry or size of the flow cell and the channel, or a combination thereof. For example, the volume of the air gap can be selected to fill approximately 30%, 40%, 50%, 60%, or 70% of the total volume of each channel. As another example, the volume of the air gap can be adjusted based on the reagent to be subsequently applied; for example, the air gap can be increased if higher cleanliness or reduced contamination is required.

[0242] The air gap flowing through the one or more channels can be configured to push the existing reagent in the channel toward and from the outlet. Thus, the sequencing reagent subsequently delivered can achieve high homogeneity in the flow cell. In existing flow cells that rely solely on wash buffer between sequencing reagent deliveries, mixing of sequencing reagents with wash buffer or liquid is unavoidable, and a concentration gradient of sequencing reagents may exist, with a higher concentration at one end near the landing region or inlet and a lower concentration at the opposite end near the outlet. Such gradients or heterogeneity can be gradually reduced by repeated washing, but are still difficult to eliminate completely. Gradients or heterogeneity of reagent concentrations can lead to less accurate and unreliable sequencing analyses of patches toward opposite ends of the flow cell, at least in part due to heterogeneous reactions or attachments of compounds in the reagents to the communities or combinations thereof. Additionally, introducing air bubbles into existing flow cells between reagents can damage the channel coating or the communities or combinations thereof tethered thereon and being imaged, thereby affecting the sequencing process. The flow cell device described herein can advantageously utilize an air gap between sequencing reagent applications to minimize or eliminate reagent concentration gradients or heterogeneity along the y-axis in the flow cell with minimal or no damage to the sample tethered thereon during sequencing.

[0243] In some embodiments, the air gap and wash solution can be combined to achieve optimal channel cleaning. In some embodiments, an air gap may be used alone to achieve optimal channel cleaning. In some embodiments, a washing scheme using an air gap, a washing solution, or both may be determined based on the contamination level of the reagent to be delivered. In some embodiments, a washing scheme using an air gap, a washing solution, or both may be determined based on the cost of the reagent, together with or in combination with other factors such as the contamination level. In some embodiments, when an air gap and a washing solution are used in combination in a washing scheme, the order in which they are used may differ. The air gap may be applied after or before any number of rinses with the washing solution. In some embodiments, it may be applied in any selected...The air gap is purged between washes with a certain amount of washing solution.

[0244] The air gap flowing through the one or more channels may dry the coating of the one or more channels, but the function of the coating may remain unchanged after the one or more air gaps have flowed through them. In some embodiments, the air gap flowing through the one or more channels may dry the colonies tethered to the channel coating. However, the air gap does not damage the colonies and ensures that the colonies can undergo proper sequencing reactions when subsequent liquid reagents are rinsed through the channels. Therefore, flow cell devices with such channels can be cleaned by purging the channels with air gaps alone or in combination with reagent washing. Using air gap cleaning can improve the efficiency and effectiveness of cleaning the channels while reducing the reagent costs required for washing and performing sequencing analysis, while meeting predetermined contamination requirements.

[0245] In some embodiments, the surface of the first coating 522a may be passivated. In some embodiments, the surface is passivated with the first coating 522a, which is used to immobilize the surface-capturing primers, nucleic acid template molecules, or both, to capture polynucleotides thereon. In some embodiments, the surface may contain the polynucleotides captured thereon during sequencing. In some embodiments, the polynucleotides captured thereon are configured for imaging in a sequencing cycle.

[0246] In some embodiments, a first coating 522a of the surface comprises one or more hydrophilic polymer coatings. The first coating may comprise a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating. The hydrophilic polymer coating may comprise PEG. The hydrophilic polymer layer may comprise a branched hydrophilic polymer, and the branched hydrophilic polymer may comprise at least 8 branches. In some embodiments, the water contact angle of the hydrophilic polymer coating does not exceed about 50 degrees.

[0247] In some embodiments, the surface comprises at least one discrete region comprising a plurality of cloned amplified sample nucleic acid molecules that have been annealed to a plurality of attached oligonucleotide molecules. In some embodiments, at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a tandem polymer that has been annealed to at least one of the plurality of attached oligonucleotides.

[0248] In some embodiments, at least one of the plurality of sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule, the single-stranded multimeric nucleic acid molecule comprising a repeating sequence of regularly occurring monomeric units. The length of the single-stranded multimeric nucleic acid molecule may be at least 10 kilobases. In some embodiments, at least one of the plurality of sample nucleic acid molecules further comprises a double-stranded monomeric copy of a regularly occurring monomeric unit. The plurality of oligonucleotide molecules may be present at various locations on the surface with a substantially uniform surface density. The plurality of oligonucleotide moleculesThe oligonucleotide molecules may exist at a first location on the surface with a local surface density of at least about 100,000 molecules / μm², and at a second location on the surface with a second local surface density. In some embodiments, the plurality of oligonucleotide molecules exist at a surface density of at least about 1,000 molecules / m².

[0249] In some embodiments, the first coating may comprise a plurality of hydrophilic polymer coatings. The first coating may comprise a first layer comprising a monolayer of polymer molecules tethered to the surface of the substrate. The first coating may further comprise a second layer comprising a second monolayer of polymer molecules tethered to polymer molecules of the first layer; and a third layer comprising a third monolayer of polymer molecules tethered to polymer molecules of the second layer, wherein at least one of the first, second, or third layers comprises branched polymer molecules.

[0250] In some embodiments, the third layer may comprise oligonucleotides tethered to polymer molecules of the third layer. The oligonucleotides tethered to polymer molecules of the third layer may be distributed at multiple depths throughout the third layer. Specification page 38 / 105, page 61, CN 121057626 A

[0251] In some embodiments, the first coating may comprise a fourth layer comprising branched polymer molecules tethered to polymer molecules in a third layer; and a fifth layer comprising polymer molecules tethered to branched polymer molecules in the fourth layer. In some embodiments, the polymer molecules in the fifth layer further comprise oligonucleotides tethered to the polymer molecules in the fifth layer. The oligonucleotides tethered to the polymer molecules in the fifth layer are distributed at multiple depths throughout the fifth layer.

[0252] In some embodiments, the hydrophilic polymer coating of the first coating may comprise molecules selected from the group consisting of: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.

[0253] In some embodiments, when the cloned and amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine dye-3, the ratio of the fluorescence intensity of the cloned and amplified cyanine dye-3 labeled sample nucleic acid molecule or its complementary sequence to the fluorescence intensity of the nonspecific cyanine dye-3 adsorption background (B gap) shown in the surface image is at least 3:1.

[0254] In some embodiments, the ratio of fluorescence intensity of the cloned, anthocyanin-3-labeled sample nucleic acid molecule or its complementary sequence shown in the surface image to the fluorescence intensity of the combination of nonspecific anthocyanin-3 dye adsorption background and nonspecific amplification background (B-gap + B-endoplasm) is at least 3:1.

[0255] In some embodiments, when the cloned, anthocyanin-3-labeled sample nucleic acid molecule or its complementary sequence is labeled with anthocyanin-3, the ratio of fluorescence intensity of the cloned, anthocyanin-3-labeled sample nucleic acid molecule or its complementary sequence shown in the surface image to the fluorescence intensity of the nonspecific dye adsorption background (B-gap) is at least 5:1.

[0256] In some embodiments, the ratio of fluorescence intensity of the cloned, anthocyanin-3-labeled sample nucleic acid molecule or its complementary sequence shown in the surface image to the fluorescence intensity of the combination of nonspecific anthocyanin-3 dye adsorption background and nonspecific amplification background (B-gap + B-endoplasm) is at least 5:1.

[0257] In some embodiments, when the cloned and amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine dye-3, the fluorescence image of the surface is acquired under non-signal saturation conditions using an inverted microscope equipped with a 20× objective lens, NA = 0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for cyanine dye-3 emission, and a camera while the surface is immersed in buffer, the contrast-to-noise ratio (CNR) of the fluorescence image is at least 20.

[0258] In some embodiments, one or more inner surfaces of the inner surface 521a may be coated with a third coating (not shown) of fluorescent beads in combination with the first coating 522a.

[0259] The fluorescent beads may be chemically immobilized to the surface. The fluorescent beads may be covalently immobilized to the surface. The fluorescent beads may be immobilized to or fixedly attached to the surface by forming a coating, such as a third coating, on the surface so that the fluorescent beads remain fixed or immobilized relative to the surface 521a. The coating may be applied directly to the surface 521a and in contact with the surface. Alternatively, the third coating may be applied indirectly to surface 521a or may not be in direct contact with said surface. In some embodiments, the third coating may be applied between surface 521a and the first coating 522a.

[0260] In some embodiments, fluorescent beads are chemically immobilized to the surface. In some embodiments, fluorescent beads are covalently immobilized to the surface. In some embodiments, fluorescent beads are pre-activated to enable them to attach to the surface chemically. In some embodiments, fluorescent beads are pre-activated to enable them to attach covalently to the surface. In some embodiments, sequencing system 110 is used to simultaneously image polynucleotide clusters or communities captured thereon and fluorescent beads in one or more sequencing cycles.

[0261] In some embodiments, reagents can be applied to the flow cell device via channels to improve the wettability of samples on surfaces, such as cultured cells or tissues. In some embodiments, such reagents may include various buffers, such as PBS buffer, for sample preparation as described in the DNA sequencing sample instruction manual (page 39 / 105, 62 CN 121057626 A). In some embodiments, such reagents may include various surfactants. In some embodiments, reagents can be applied to the flow cell device to increase the wettability of samples on a surface by 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 80%, 100%, 120%, 150%, 180%, 200%, or more, compared to the wettability prior to the application of such reagents. In some embodiments, reagents can be applied to the flow cell device via microfluidic channels to reduce the surface tension at air / liquid interfaces (e.g., air / liquid interfaces of bubbles). In some embodiments, reagents may be applied to the flow cell device to reduce the surface tension by 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 80%, 100%, 120%, 150%, 180%, 200%, or more compared to the surface tension before reagent application. In some embodiments, reagents may be applied to the flow cell device to increase the bubble size by 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 80%, 100%, 120%, 150%, 180%, 200%, or more compared to the bubble size before reagent application. In some embodiments, reagents to improve sample wettability may be applied during sample preparation. In some embodiments, reagents to improve sample wettability may be applied during flow cell assembly, for example, before assembling the one or more substrates into the flow cell device. Flow cell assembly may include generating a flow cell device on which the sample to be sequenced is immobilized during a sequencing run. In some embodiments, reagents to improve sample wettability may be applied during rolling circle amplification (RCA). In some embodiments, a reagent to improve sample wettability may be applied before the start of a sequencing run to produce sequencing results. In some embodiments, a reagent to reduce the surface tension of the air / liquid interface (e.g., the air / liquid interface of a bubble) may be applied during sample preparation. In some embodiments, a reagent to reduce the surface tension of the air / liquid interface (e.g., the air / liquid interface of a bubble) may be applied before flow cell assembly. In some embodiments, a reagent to reduce the surface tension of the air / liquid interface (e.g., the air / liquid interface of a bubble) may be applied during rolling circle amplification (RCA). In some embodiments, a reagent to reduce the surface tension of the air / liquid interface may be applied before the start of a sequencing run to produce sequencing results.

[0262] In some embodiments, the reagent flow rate during sample preparation can be in the range of 1 μL / sec to 5000 μL / sec. In some embodiments, the reagent flow rate during sample preparation can be in the range of 10 μL / sec to 1000 μL / sec. In some embodiments, the reagent flow rate during sample preparation can be in the range of 10 μL / sec to 500 μL / sec. In some embodiments, the reagent flow rate during sample preparation can be in the range of 20 μL / sec to 500 μL / sec. In some embodiments, the reagent flow rate during sample preparation can be within a range such that the shear stress on the sample and / or reagent can be increased by 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 80%, 100%, 120%, 150%, 180%, 200%, 250%, 200%, 350%, 400%, 450%, 500%, 600%, or more compared to the shear stress at a previous reagent flow rate. In some embodiments, the reagent and / or sample can flow at a rate within a predetermined flow rate range during rolling circle amplification (RCA).

[0263] In some embodiments, a negative pressure is applied to the flow cell apparatus, and more specifically to the lumen of the channel. The negative pressure may be applied during sample preparation and / or RCA. The negative pressure may be applied before the start of the sequencing run to produce sequencing results. In some embodiments, the negative pressure may be in the range of -1 kPa to -450 kPa. In some embodiments, the negative pressure may be in the range of -10 kPa to -350 kPa. In some embodiments, the negative pressure may be in the range of -10 kPa to -150 kPa. In some embodiments, an increase in shear stress may be obtained by raising the temperature to a temperature range of 30°C to 80°C and applying a negative pressure in the range of -10 kPa to -150 kPa.

[0264] In some embodiments, reagents and / or samples may be heated for a predetermined duration in the temperature range of 30°C to 80°C during sample preparation, such that the shear stress on the sample and / or reagents may increase by 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 80%, 100%, 120%, 150%, 180%, 200%, 250%, 200%, 350%, 400%, 450%, 500%, 600%, or more compared to the shear stress before heating of the reagents and / or samples. In some embodiments, reagents and / or samples may be heated during rolling circle amplification (RCA).

[0265] Open landing area specification 40 / 105 pages 63 CN 121057626 A Flow cell apparatus and system may include one or more open landing areas 241, 341a, 441a, 541a,741a. Figures 30-32 and 33A-33F illustrate a flow pool apparatus having an open landing area for one or more channels.

[0266] The open landing area may be part of an inlet. The open landing area may be located on a bottom substrate. The open landing area may be fluidly connected to its corresponding channel. The open landing area may be fluidly connected to a manifold or connector. Open landing areas 341a, 441a, 541a may be part of inlets 340a, 440a, 540a. Open landing areas 341a, 441a, 541a may be located on bottom substrates 330a, 430a, 530a. Open landing areas 341a, 441a, 541a may be fluidly connected to their corresponding channels 350a, 450a, 550a.

[0267] The inlet may include a void or hole in the top substrate located above at least a portion of the open landing regions 241, 341a, 441a, 541a, 741a. An air gap or liquid reagent, or a combination thereof, may be introduced through the void or hole of the inlet to reach the open landing region and then transferred from the open landing region to a corresponding channel. In some embodiments, the cross-sectional area of ​​the void or hole in the x-y plane may be substantially the same as or equal to the area of ​​the open landing region. In some embodiments, the cross-sectional area of ​​the void or hole in the x-y plane may be larger than the area of ​​the open landing region. In some embodiments, the void or hole may be considered to have a rectangular cross-section in the x-y plane, which is as wide as the flow cell device along the x-axis. Inlets 340a, 440a, 540a may include a void or hole in the top substrate 320a, 420a, 520a located above at least a portion of the open landing regions 341a, 441a, 541a. A gaseous or liquid reagent, or a combination thereof, can be introduced through the gaps or holes of inlets 340a, 440a, 540a to reach open landing areas 341a, 441a, 541a, and then transferred from open landing areas 341a, 441a, 541a to corresponding channels 350a, 450a, 550a. In some embodiments, the cross-sectional area of ​​the gap or hole in the x-y plane can be substantially the same as or the same as the area of ​​the open landing area, for example, as shown in Figures 31-32, 33A and 33F. In some embodiments, the cross-sectional area of ​​the gap or hole in the x-y plane can be larger than the area of ​​the open landing area, for example, in Figure 33E. The gap or hole in Figure 33E can be considered to have a rectangular cross-section in the x-y plane, which is as wide as the flow cell device along the x-axis.

[0268] The inlets and open landing areas can advantageously enable the open application of liquids or gases to the flow cell device. Inlets 340a, 440a, and 540a and open landing areas 341a, 441a, and 541a can advantageously facilitate the loading of liquids or gases into the flow pool.Open application of the flow cell device described herein, through open landing regions, advantageously removes a series of closed or locked channels, thereby significantly reducing system complexity and cost, and allowing the system and device to be more flexible in adapting to a variety of sequencing applications. Open application through open landing regions also advantageously improves the compatibility of fluid control and fluid dispensing with different flow cell devices without requiring changes to the closed channels in existing sequencing systems. For example, each dispensing tip can be used only for the corresponding reagent without contaminating other reagents. As another example, multiple dispensing tips can be used to simultaneously apply the same or different reagents to different channels to improve sequencing efficiency and reduce sequencing time.

[0269] In different embodiments, the size and shape of the pores or voids, as well as the size and shape of the open landing regions, can vary. The size and shape can be determined based on parameters in a specific sequencing application, such as flush volume, contamination threshold, flow cell size (e.g., width of flow cell channels) or dispenser parameters (e.g., size of dispensing tips). As a non-limiting example, the pores or voids are cylindrical, with walls extending along the z-axis and orthogonal to the substrate. As a non-limiting example, the aperture or void is cylindrical, as shown in FIG33C, with its walls extending along the z-axis and orthogonal to the substrate. However, the shape, size, or combination thereof of the aperture or void can vary. For example, the aperture or void can have an inverted conical shape, with a wider opening at the top that tapers toward the channel to reduce reagent residue that may remain in the inlet. In some embodiments, a larger open landing area can better facilitate reagent transfer into the channel, and maintaining the size of the open landing area within a predetermined ratio to the width of the channel can also better facilitate reagent transfer into the channel. As a non-limiting example, the diameter of the open area, for example, the widest dimension in the x-y plane, can range from about 3 mm to about 40 mm. In some embodiments, the diameter of the open area is approximately the same as the width of the corresponding channel. In some embodiments, the diameter of the open area is about 10%, 20%, 30%, 40%, or 50% smaller than the width of the corresponding channel. As a non-limiting example, the diameter of the orifice or void, for example, the widest dimension in the x-y plane, can range from about 3 mm to about 40 mm. In some embodiments, the diameter of the orifice or void is approximately the same as the width of the corresponding channel. In some embodiments, the diameter of the orifice or void is about 10%, 20%, 30%, 40%, or 50% smaller than the width of the corresponding channel.

[0270] To work with an open landing area, the flow cell system may include a fluid control device that may include a dispenser configured to openly dispense one or more reagents to the inlet.Working in conjunction with open landing areas, the flow cell system may include a fluid control device that may include dispensers 280a, 580a configured to openly dispense one or more reagents into inlet 540a. The dispenser may openly dispense from its tip into the open landing area through a gap or orifice in the inlet. The dispenser may openly dispense from its tip into open landing areas 341a, 541a through a gap or orifice in the inlet. In some embodiments, there is no conduit connecting the dispenser and the inlet. In some embodiments, the dispenser directly contacts a portion of the inlet, such as the landing area or a gap wall, to openly dispense reagents. In some embodiments, the dispenser does not directly contact any physical portion of the inlet, but its tip may extend into a gap or orifice in the inlet. In some embodiments, at least a portion of the tip of the dispenser contacts the open landing area. In some embodiments, the tip of the dispenser does not directly physically contact the open landing area.

[0271] The dispenser may include more than one dispensing tip, such as a pipette tip, such that each different reagent can have its own dispensing tip without reagent mixing occurring in the dispenser or dispensing tip. In a sense, the dispenser disclosed herein removes shared tubing from existing flow cell systems and reduces dead volume in shared tubing, allowing for a significant reduction in reagent consumption required for the same sequencing process. Additionally, removing shared tubing and using separate dispensing tips reduces reagent mixing and resulting contamination of reagents dispensed into the flow cell apparatus.

[0272] In some embodiments, the dispenser and its tips can be manually operated to move or dispense, or a combination thereof. In some embodiments, the dispenser and its tips can be automatically operated to move or dispense, or a combination thereof. For example, the dispenser may include an array of dispensing tips, each dispensing tip in fluid communication with a reagent reservoir in the cartridge, and a robotic arm moves the array to position a corresponding tip above a landing area and then controls the dispensing. When the next reagent needs to be delivered, the robotic arm can retract a previous dispensing tip and position the next reagent tip in the array for dispensing. Automated operation of the dispenser and its tips can be controlled by executable software on the hardware processor of the sequencing system described herein. In some embodiments, multiple dispensing tips can be controlled to dispense simultaneously. In some embodiments, the same dispensing tip can be controlled to dispense to a first open landing area and then move to a second open landing area for dispensing.

[0273] In some embodiments, the open landing area may be associated with the risk of contamination by the external environment (e.g., dust, fibers, and debris). Contaminants may enter the microfluidic channel from the open landing area. When contaminants enter the microfluidic channel, they may remain in the microfluidic channel and cause surface, flow patterns, and liquid exchange efficiency to deteriorate.Changes in the flow cell apparatus can reduce sequencing quality in areas near contaminants, such as fibers.

[0274] In some embodiments, the flow cell apparatus may include one or more filters configured to capture or trap contaminants that may otherwise enter the microfluidic channel. The filters may be installed at different locations between the open landing area and the microfluidic channel. Figures 43A-43B illustrate exemplary embodiments of the filters. In some embodiments, filters are installed to capture contaminants from the environment. Filters can advantageously facilitate the capture of contaminants, i.e., any unwanted particles or residues from the external environment entering the microfluidic channel. Filters can facilitate the avoidance of spatial closure of the surface of the microfluidic channel and prevent changes in the flow pattern through the microfluidic channel to ensure efficient liquid exchange, thereby allowing accurate and reliable sequencing reactions.

[0275] In some embodiments, the filters may be configured to capture or trap solid contaminants. In some embodiments, the filters may be configured to capture or trap air bubbles, for example, within a diameter limitation range. In some embodiments, filter installation does not alter the hydrodynamics in the microfluidic channel because flow circulation can be optimized, such as the flow rate, velocity, etc., of the installed filter to achieve the same desired flow dynamics prior to filter installation. In some embodiments, the filter does not change the average flow rate in the microfluidic channel but may alter the local flow field in the vicinity. In some embodiments, sequencing reactions adjacent to the filter may or may not take into account the sequencing results.

[0276] In some embodiments, the filter may comprise a variety of materials. In some embodiments, the filter may comprise one or more of the same materials used to construct the flow cell device. In some embodiments, the filter may comprise one or more of the following: glass, plastic, polymer, and hydrogel. In some embodiments, the filter may comprise one or more microfabricated materials.

[0277] In some embodiments, the filter may comprise more than one filter 1012 positioned in a 3D pattern, for example, as shown in FIG43B. In some embodiments, the filter may be installed at different locations along the fluid pathway on the FC device, through which sequencing reagents may pass after reaching the open landing area and before leaving the flow cell device. In some embodiments, different filters may be installed at different locations on the flow cell device. For example, a first filter may be installed near the open landing area as shown in FIG43A-43B. A second additional filter may be installed at or near the center of the microfluidic channel to the right of the first filter (not shown).

[0278] In some embodiments, the flow cell device 112 further includes cleaning outlets 470a, 570a, and 770a. Cleaning outlets470a, 570a, and 770a may be located in one or more of the substrates, such as the bottom substrates 430a and 530a. In some embodiments, cleaning outlets 470a, 570a, and 770a may be located as side ports (not shown) on the top substrate or in the middle substrate. In some embodiments, the side ports extend at least along a direction perpendicular to or nearly perpendicular to the y-direction. In some embodiments, the side ports extend at least along a direction perpendicular to or nearly perpendicular to the z-direction. In some embodiments, the side ports extend at least along a direction inclined to the x-direction. In some embodiments, the side ports extend at least along a direction inclined to the y-direction. In some embodiments, the side ports extend at least along a direction inclined to the z-direction. In some embodiments, the side ports extend at least along a direction perpendicular to or nearly perpendicular to the x-y plane. In some embodiments, the side ports extend at least along a direction perpendicular to or nearly perpendicular to the x-z plane. In some embodiments, the side ports extend at least along a direction perpendicular to or nearly perpendicular to the x-y plane. In some embodiments, the side ports extend at least along a direction perpendicular to or nearly perpendicular to the y-z plane. In some embodiments, the side ports extend at least along a direction inclined to the x-y plane. In some embodiments, the side port extends at least along a direction inclined to the x-z plane. In some embodiments, the side port extends at least along a direction inclined to the y-z plane.

[0279] Cleaning outlets 470a, 570a, and 770a may be fluidly connected to inlets 440a and 540a. In some embodiments, cleaning outlets 470a, 570a, and 770a are configured to be connected to a fluid drive device, such as a pump or vacuum device 471a of a fluid control device. Pump 471a may be a supplement to pump 472a connected to outlet 460a. In some embodiments, the same fluid drive device, such as a pump, may be connected to outlets 460a, 560a and cleaning outlet 470a.

[0280] The distance from cleaning outlets 470a, 570a, and 770a to inlets 440a and 540a may be shorter than the distance to outlets 460a and 560a. The distance may be in the x-y plane. The shorter distance from the clean outlet to the inlet is designed to facilitate the transfer of liquid or gas from the open landing area to the clean outlet.

[0281] In some embodiments, the relative positions of the clean outlets 470a, 570a, 770a to the inlets 440a, 540a, 740a may differ. In some embodiments, the clean outlet 770a may be located directly below the open landing area 741a, for example, in the specification on pages 43 / 105, 66 CN 121057626 A 38A-38E and 39C. In such embodiments, the clean outlet 770a is directly connected to the open landing area.

[0282] In some embodiments, the cleaning exits 470a and 570a may not be directly located below the open landing area, but may be located at a distance from the open landing area, for example, in Figures 32, 33A, 37A-37E, and 39A-39B. In such embodiments, the cleaning exits 470a and 570a are not directly connected to the corresponding open landing areas, but are connected via tapered transition portions 454a and 554a therebetween.

[0283] The distance from the cleaning exit to the nearest edge or center of the open landing area may be 0 mm or about 0 mm. When the cleaning exit is not directly located below the open landing area, the distance from the cleaning exit to the nearest edge or center of the open landing area may be about 0 mm to about 20 mm. When the cleaning exit is not directly located below the open landing area, the distance from the cleaning exit to the nearest edge or center of the open landing area may be about 0 mm to about 15 mm. When the cleaning exit is not directly located below the open landing area, the distance from the cleaning exit to the nearest edge or center of the open landing area may be about 0 mm to about 10 mm. When the clean exit is not directly below the open landing area, the distance from the clean exit to the nearest edge or center of the open landing area can be from about 3 mm to about 10 mm.

[0284] When the clean exit is not directly below the open landing area, the distance from the clean exit to the nearest edge or center of the open landing area can be from 0 mm to 15 mm. When the clean exit is not directly below the open landing area, the distance from the clean exit to the nearest edge or center of the open landing area can be from 0 mm to 10 mm. When the clean exit is not directly below the open landing area, the distance from the clean exit to the nearest edge or center of the open landing area can be from 3 mm to 10 mm.

[0285] In some embodiments, reagent residues, such as menisci, as shown in the bottom inset of FIG32, may remain on the walls of the orifice of the open landing area or the inlet, or a combination thereof. If such residues are not removed, unintentional mixing may occur when subsequent reagents are delivered to the open landing area, and thus contaminate the sequencing reaction in the channel. Liquid washing alone may not be effective in removing residual reagents such as those on the meniscus, thus requiring multiple rinses to completely remove residues from existing flow cell systems, increasing washing time and costs. Fluidly connected cleaning outlets 470a and 570a can advantageously facilitate the time- and cost-effective removal of such residues. In some embodiments, mechanical driving force can be applied through the cleaning outlet, for example by a pump or inlet vacuum device, to completely remove such residual reagents from open landing areas. Therefore, the time and washing volume required to remove residues to achieve satisfactory contamination levels can be effectively improved from existing flow cell arrangements.

[0286] The size and shape of the cleaning outlet can be customized to suit different sequencing applications. Although the cleaning outlet is shown as a cylinder in Figure 33C, it can be made into different shapes, such as cones, inverted cones, etc. In some embodiments, the size and shape of the cleaning outlet can be the same as the size and shape of the outlet. In some embodiments, the size of the cleaning outlet can differ from the size of the outlet by no more than about 10%, 20%, or 30%. In some embodiments, the diameter of the cleaning outlet in the x-y plane is about 0.3 mm to about 10 mm. In some embodiments, the height of the cleaning outlet in the z-direction is the same as the height of the bottom substrate. In some embodiments, the height of the cleaning outlet is about 0.3 mm to about 3 mm. In some embodiments, the height of the cleaning outlet is about 0.5 mm to about 1 mm. In some embodiments, the diameter of the cleaning outlet in the x-y plane is 0.3 mm to 10 mm. In some embodiments, the height of the cleaning outlet in the z-direction is the same as the height of the bottom substrate. In some embodiments, the height of the cleaning outlet is 0.3 mm to 3 mm. In some embodiments, the height of the cleaning outlet is 0.5 mm to 1 mm.

[0287] In some embodiments, a portion of the substrate, in addition to the inner surface of the channel, may be covered with a second coating, such as a smoothing coating, to facilitate fluid transfer on the coating, said coating being alone or in combination with the first coating disclosed herein. The second coating may be different from the first coating of the channel. The second coating may be applied directly to a substrate without the first coating applied (see specification page 44 / 105, 67 CN 121057626 A). The second coating may be applied to a substrate on top of which the first coating is applied.

[0288] The thickness of the coating along the z-axis may be customized such that it does not interfere with or reduce the fluid flow rate or other fluid parameters or combinations thereof of the channel compared to a flow pool device without a coating. The thickness of the coating along the z-axis may be customized such that it increases or promotes the fluid flow rate or other fluid parameters or combinations thereof of the channel compared to a flow pool device without a coating.

[0289] In some embodiments, the open landing area is covered with a coating. In some embodiments, the coating may be applied to at least a portion of the open landing area. In some embodiments, the coating may be applied to any combination of surfaces of the substrate, in addition to the inner surface of the lumen defining the channel. The coating can effectively facilitate the transfer of liquid from the open landing area to the channel and / or clean outlet to exit the flow cell device. For example, when reagent is transferred into the channel, the coating can help reduce the volume of residual reagent on the open landing area. As another example, when an inlet vacuum force is applied through the clean outlet, the coating can facilitate the complete removal of residual reagent on the open landing area.

[0290] In some embodiments, the open landing area 341a is covered with a second coating 342a. Figure 31 illustrates the open landing area...An embodiment of a second coating 342a on region 341a, the remainder of open landing region 343a, and a portion of the top substrate above open landing region 343a. A small right-hand view of Figure 31 shows a schematic diagram of the second coating 342a having reagent droplets thereon. In some embodiments, the second coating 342a may be applied to at least a portion of open landing region 341a. In some embodiments, the second coating 342a may be applied to any combination of surfaces of the substrate, except for the inner surface of the lumen defining the channel. The second coating 342a can effectively facilitate the transfer of liquid from open landing region 341a to channels 350a, 550a, or cleaning outlet 570a to exit the flow cell apparatus. For example, when reagent is transferred into the channel, the second coating 342a can help reduce the volume of residual reagent on the open landing region. As another example, when an inlet vacuum force is applied through the cleaning outlet, the second coating 342a can facilitate the complete removal of residual reagent on the open landing region.

[0291] In some embodiments, the coating may be a variety of liquid-repellent coatings. In some embodiments, the coating may be a superhydrophobic coating. In some embodiments, the coating comprises a smooth superhydrophilic covalently attached liquid (SOCAL) coating. In some embodiments, the coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates. In some embodiments, the coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers. The one or more brine monomers may comprise dimethyldimethoxysilane (PDMS). In some embodiments, the low surface energy of the one or more brine monomers may be below about 10 mJ / m2, 15 mJ / m2, 20 mJ / m2, 25 mJ / m2, or 20 mJ / m2.

[0292] In some embodiments, the second coating 342a may be any liquid-repellent coating. In some embodiments, the second coating may be a superhydrophilic coating. In some embodiments, the second coating comprises a smooth superhydrophilic covalently attached liquid (SOCAL) coating. In some embodiments, the second coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates. In some embodiments, the second coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers. The one or more brine monomers may comprise dimethyldimethoxysilane (PDMS). In some embodiments, the low surface energy of the one or more brine monomers may be below about 10 mJ / m², 15 mJ / m², 20 mJ / m², 25 mJ / m², or 20 mJ / m².

[0293] The coating or second coating may be formed using various methods. For example, it can be formed by impregnating a lubricant with a...The coating is formed in one or more porous surfaces. In some embodiments, the coating comprises a smooth liquid-filled porous surface (SLIPS). In some embodiments, the lubricant comprises a liquid with low surface energy, wherein the low surface energy is below a predetermined threshold. The predetermined threshold may be about 20 millijoules per square meter (mJ / m2). In some embodiments, the predetermined threshold may be about 10 mJ / m2, 12 mJ / m2, 14 mJ / m2, 16 mJ / m2, 18 mJ / m2, 20 mJ / m2, 22 mJ / m2, 24 mJ / m2, 26 mJ / m2, 28 mJ / m2, or 30 mJ / m2. In some embodiments, the lubricant comprises silicone oil. In some embodiments, the coating comprises a low surface energy of about 10 mJ / m2, 15 mJ / m2, 20 mJ / m2, 25 mJ / m2, or 20 mJ / m2.

[0294] Contamination Levels and COGS Savings In some embodiments, cleaning of the flow cell apparatus can be achieved by using, alone or in different combinations, one or more of the following: an open landing area with open distribution, a channel coating, a smooth coating of the open landing area, and a clean outlet and a vacuum. Figures 30-32, 33A-33F illustrate non-limiting embodiments of combinations of one or more of these in the flow cell apparatus.

[0295] Figure 35 shows the contamination levels of the flow cell apparatus disclosed herein compared to existing flow cell apparatuses. Images of the flow cell channels are obtained by flushing volume. The flushing volume in this embodiment is approximately 60 microliters (μL), determined at least based on the channel size and geometry. The average contamination rate of the flow cell channels for all three flow cell apparatuses and two existing flow cells is approximately 1%. As the flushing volume increases, the contamination level begins to decrease. When the flushing factor reaches 5, the total volume of the flushing reagent is approximately 300 μL. At this rinsing factor, the three flow cell devices disclosed herein (with SLIPS coating on open landing readouts, inlet vacuum devices with clean exits, or combinations thereof) exhibit contamination levels below 0.01%, while existing flow cells show significantly higher contamination levels, exceeding 0.1%. Existing flow cell devices require more than 10 rinsing factors or more than 600 μL of washing reagent to achieve similar contamination levels to the three flow cell devices disclosed herein. In this specific embodiment, using more than 300 μL of rinsing does not further reduce the contamination level to a significant level. A contamination level of approximately 0.001% is satisfactory for all reagents used in NGS sequencing applications. The three flow cell devices advantageously achieve contamination levels for accurate and reliable sequencing processes, significantly reducing the cost of goods sold (COGS) compared to existing methods.

[0296] Figure 42 illustrates the average residual or contamination levels between different patches of the flow cell apparatus disclosed herein. Variations in contamination across the flow cell apparatus can be caused by the spatial positioning of the patch within the flow cell and its relative position to the inlet or outlet, or a combination thereof. The average contamination level of different patches in the flow cell apparatus disclosed herein is effectively reduced to less than 1% within the first wash volume cycle. By the third wash volume or cycle, the residual or contamination levels of different patches are reduced to below 0.001%. By the third wash volume, the average patch contamination is below 0.001%. By the third or fourth wash volume, the contamination of a single patch across the flow cell is below 0.001%. The wash volume is approximately 60 μL, ensuring that the contamination level of a single patch is reduced to below 0.001% regardless of its spatial positioning within the flow cell, with a total wash volume of reagents or wash solution of 240 μL.

[0297] In one embodiment, by using the flow cell apparatus disclosed herein, the volume of affinity sequencing reagents required for stepping, cutting, and imaging is significantly reduced. The stepping reagent requires a volume of approximately 430 μL and is reduced to approximately 90 μL by active volume reduction (AVR) to recover a portion of the reagent. AVR can be used in both existing flow cell systems and the flow cell system disclosed herein. AVR can be approximately 40%, 50%, 60%, 70%, 80%, or 90% of the total volume required for the sequencing application. The total volume can be the volume without AVR. AVR saves approximately 5 times the reagent compared to existing flow cell devices. Without AVR, it still reduces by approximately 2.5 times compared to existing flow cell devices. In some embodiments, with AVR, the cleavage, capture, and imaging reagent is reduced from approximately 300 μL to approximately 60 μL. Table 1 below shows the volume of sequencing reagent required using existing flow cell systems and the volume of reagent saved or reduced by COGS required using the flow cell device disclosed herein.

[0298] Table 1: Reagent consumption reduction is achieved during the same sequencing application by the flow cell system disclosed herein compared to existing flow cell systems. Specification 46 / 105 pages 69 CN 121057626 A

[0299] Fluid Control Device This document discloses a fluid control device that can be coupled to a flow tank device and actively applies mechanical forces to dispense or collect liquids or gases or combinations thereof from the flow tank device.

[0300] In some embodiments, the fluid control device may include a pump, a vacuum device, or any other device that can actively apply mechanical forces to the lumen of a channel or an open landing area or a combination thereof through an outlet or a clean outlet. Figure 32 shows a fluid control device having a vacuum device 472a coupled to all outlets 460a of the flow tank device 4112. Figure 32Another vacuum device 471a is shown, connected to the clean outlet 470a of the flow cell device 112. Vacuum devices 471a and 472a may be the same vacuum device or pump.

[0301] In some embodiments, the fluid control device may include dispensers 280a, 580a having one or more dispensing tips. Dispensers 280a, 580a may dispense a preset amount of reagent into the inlet within a certain time window.

[0302] In some embodiments, the fluid control device may include a robotic arm that controls the movement of the dispensers. In some embodiments, the robotic arm may move the dispensers in 3D space such that the dispensing tips may reach a specific position before they begin dispensing. In some embodiments, the robotic arm may retract the dispensing tips after one dispensing and move a second dispensing tip to a position for subsequent dispensing.

[0303] In some embodiments, the fluid control device may include a dispensing roller configured to dispense reagent, as shown in FIG34A. The reagent can be dispensed by dispenser 680a onto a continuous track 691a that rolls on one or more wheels, and the wheels of the rollers can roll the track 691a and the reagent into an open landing area of ​​the flow cell. In this specific embodiment, the inlet can be a side port located at the edge of the substrate. An active force can be applied at the outlet to facilitate the delivery of the reagent from the track to the inlet.

[0304] In some embodiments, the fluid control device can include a dispensing plate having an electrowetting surface. As shown in FIG34B, the dispensing plate 692a can be translated, thereby translating the reagent dispensed thereon to the inlet, which in this embodiment is a side port at the edge of the substrate.

[0305] In some embodiments, the fluid control device can include a reagent reservoir and a pipette, as shown in FIG34C. In this specific embodiment, one end of the pipette 693a can be inserted into the reagent reservoir 694a, and the other end of the pipette can point towards or contact the inlet. The reagent can be drawn out in a controlled manner into the open landing area of ​​the flow cell. In this embodiment, the open landing area faces downward, and the orifice or gap of the inlet is located in the bottom substrate. Various mechanisms can be used to control the sipping action. For example, an active mechanical force can be applied from the outlet to draw a predetermined amount of reagent from the reservoir. Different pipettes can be used for different reagents to avoid unintentional mixing of reagents in pipette 693a.

[0306] Method This document discloses a method for using the flow cell device 112 to perform or facilitate sequencing analysis or a combination thereof using a sequencing system 110. This document also discloses a method for manufacturing the flow cell device 112, which can be used to perform or facilitate sequencing analysis or a combination thereof. The methods herein may include some or all of the operations disclosed herein.Operations may be performed in, but are not limited to, the order described herein.

[0307] Operations herein may be performed manually. Operations may be performed automatically by a robotic arm or the like (not shown). The robotic arm may be controlled by a computer system, such as 126 in FIG. 1, to automatically perform some or all of the operations disclosed herein. Alternatively, computer system 126, dedicated processor 118, FPGA 120, or a combination thereof may be programmed to control the robotic arm. Software, firmware, hardware, or a combination thereof may be installed on the computer system of the robotic arm, which may cause the computer system to perform the operations or actions disclosed herein during operation.

[0308] Methods may be performed by one or more processors in the computer system disclosed herein, such as 126. In some embodiments, a processor may include one or more of the following: a processing unit, an integrated circuit, or a combination thereof. For example, a processing unit may include a central processing unit (CPU) or a graphics processing unit (GPU), or a combination thereof. An integrated circuit may include a chip such as a field-programmable gate array (FPGA). In some embodiments, a processor may include a computing system. In some embodiments, some or all of the operations in the methods described herein may be performed by one or more of the following: FPGA, ASIC chip, neural processing unit (NPU), artificial intelligence chip (AI chip), tensor processing unit (TPU), graphics processing unit (GPU).

[0309] In some embodiments, some or all of the operations in the methods may be performed by an FPGA. In an embodiment, when some operations are performed by an FPGA, data after the operations are performed by the FPGA may be transferred from the FPGA to the CPU so that the CPU can use such data to perform subsequent operations in the method. Similarly, data may also be transferred from the CPU to the FPGA for processing by the FPGA. In some embodiments, all of the operations in the methods may be performed by the CPU. Alternatively, operations performed by the CPU may be performed by other processors such as dedicated processors or FPGAs. In some embodiments, all of the operations in the methods may be performed by an FPGA.

[0310] The method of manufacturing the flow cell device disclosed herein may include the operation of obtaining the one or more substrates. The operation of obtaining the one or more substrates may include obtaining the one or more substrates respectively such that the one or more substrates are not physically connected or bonded to each other.

[0311] The method disclosed herein may include the operation of generating one or more channels in the one or more substrates. In some embodiments, the channel is formed as a hole entirely within the intermediate substrate. In some embodiments, forming the channel includes forming a groove on a top or bottom substrate and forming a hole on the intermediate substrate, and the channel can be formed by stacking the groove and the hole together. In some embodiments, forming the channel involves forming a hole in each of two adjacent substrates.Grooves are formed and the grooves are combined together by etching or any other mechanism to form a channel. This disclosure does not limit the mechanisms by which holes, grooves, or cavities can be formed in a substrate. When the substrates are fixedly joined together, such as by bonding, holes, grooves, or cavities can form a channel between air gaps that allow fluid to flow through.

[0312] The methods disclosed herein may include the operation of forming an inlet. The operation of forming an inlet may include forming a hole or gap in at least one of the one or more substrates and forming an open landing area. The hole or gap may be located at or near one end of the substrate or channel or a combination thereof. For example, forming an inlet may include forming a cylindrical hole in a top substrate and forming an open landing area in an intermediate substrate that matches the positioning of the cylindrical hole, such that when the two substrates are stacked together, the hole is directly above or at least partially above the landing area.

[0313] The methods disclosed herein may include the operation of forming an outlet. The operation of forming an outlet may include forming a hole or gap in at least one of the one or more substrates. For example, forming an outlet may include forming a cylindrical hole from an inlet in a bottom substrate, the cylindrical hole being located at or near opposite ends of the substrate or channel or combination thereof. Specification 48 / 105 pages 71 CN 121057626 A

[0314] In some embodiments, the inlet and outlet are fluidly connected to the one or more channels.

[0315] The methods disclosed herein may include operations of fixing substrates together, such as bonding substrates with pressure-sensitive adhesive. The bonding operation may be achieved by chemical, mechanical or laser bonding, but is not limited to such bonding techniques.

[0316] The methods disclosed herein may include coating at least a portion of the surface of the one or more channels with a first coating as disclosed herein. The surface may be an inner surface of a lumen defining the one or more channels. For example, the surface may include a top or bottom inner surface.

[0317] The methods disclosed herein may include coating at least a portion of the surface of the one or more channels with a further coating of the first coating, such as a third coating of fluorescent beads.

[0318] The methods disclosed herein may include operations of covering at least a portion of an open landing area with a second coating as disclosed herein. The second coating may be different from or the same as the first coating in the channel. In some embodiments, the process of applying the second coating may be different from or the same as applying the first coating in the channel. In some embodiments, at least some actions in the entire process of applying the second coating may be different from or the same as applying the first coating in the channel.

[0319] In some embodiments, coating the open landing area includes impregnating a lubricant into one or more porous surfaces. In some embodiments, coating the open landing area includes acid-catalyzed graft polycondensation of one or more brine monomers.

[0320] In some embodiments, the method of manufacturing the flow cell device further includes the operation of forming a clean outlet in the one or more substrates. The operation of forming the clean outlet may include forming a clean outlet in fluid communication with an inlet, and positioning the clean outlet closer to the inlet than to an outlet. The operation of forming the clean outlet may further include forming the clean outlet in a predetermined size and shape. For example, the size and shape of the clean outlet may be substantially the same as that of the outlet. The operation of forming the clean outlet may further include forming the clean outlet in a bottom substrate, a top substrate, an intermediate substrate, or a combination thereof. As an example, th...

Claims

1. A flow cell apparatus comprising: (a) a carrier comprising one or more substrates, wherein the one or more substrates include an inlet and an outlet, wherein the inlet includes an open landing area; and (b) One or more channels defined by the one or more substrates, wherein the one or more channels are fluidly connected to the inlet and the outlet, wherein the one or more channels are configured to allow fluid or air gap flow between the fluid and another fluid through the one or more channels.

2. The flow pool apparatus of claim 1, wherein the open landing area is at least partially covered with a surface coating.

3. The flow cell apparatus of claim 1, wherein one or more channels extend from the inlet to the outlet.

4. The flow pool apparatus of claim 1, wherein the one or more channels extend along a first direction and between the inlet and the outlet.

5. The flow cell apparatus of claim 1, wherein the one or more channels are configured to allow air gap flow through the one or more channels, wherein the fluid contains a first reagent and the other fluid contains a second reagent.

6. The flow cell apparatus of claim 5, wherein the one or more channels are configured to allow air gaps to flow through the one or more channels during DNA sequencing runs.

7. The flow cell apparatus of claim 1, wherein the one or more channels are configured to allow the air gap to flow from the inlet through the one or more channels.

8. The flow cell apparatus of claim 6, wherein the one or more channels are configured to allow air gap flow through the one or more channels to facilitate reduction of contamination of the second reagent by the first reagent during the DNA sequencing run.

9. The flow cell apparatus of claim 6, wherein the one or more channels are configured to allow air gap flow through the one or more channels to reduce the minimum amount of the first reagent, the second reagent, or the washing reagent used for the DNA sequencing run.

10. The flow cell apparatus of claim 1, wherein the one or more channels comprise one or more surfaces.

11. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an inner surface.

12. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an outer surface.

13. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an inner top surface, an inner bottom surface, or both.

14. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise an outer top surface, an outer bottom surface, or both.

15. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise planar surfaces.

16. The flow cell apparatus of claim 10, wherein one or more surfaces are passivated.

17. The flow cell apparatus of claim 10, wherein one or more surfaces are passivated with a coating that immobilizes the surfaces to capture primers, nucleic acid template molecules, or both, to capture polynucleotides.

18. The flow cell apparatus of claim 17, wherein one or more surfaces comprise the polynucleotide coupled thereto.

19. The flow cell apparatus of claim 10, wherein the air gap is configured to remove moisture or liquid from at least a portion of the one or more surfaces of the one or more channels.

20. The flow cell apparatus of claim 10, wherein the air gap does not impair the chemical function of the one or more surfaces.

21. The flow cell apparatus of claim 17, wherein the coating on one or more surfaces comprises at least one hydrophilic polymer coating.

22. The flow cell apparatus of claim 17, wherein the coating on one or more surfaces comprises a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating.

23. The flow cell apparatus of claim 10, wherein the one or more surfaces comprise at least one discrete region, the at least one discrete region comprising a plurality of cloned amplified sample nucleic acid molecules, the plurality of cloned amplified sample nucleic acid molecules having been annealed to a plurality of attached oligonucleotide molecules.

24. The flow cell system apparatus of claim 21, wherein the water contact angle of the at least one hydrophilic polymer coating does not exceed about 50 degrees.

25. The flow cell apparatus of claim 23, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a tandem polymer, the tandem polymer being annealed to at least one of the plurality of attached oligonucleotide molecules.

26. The flow cell apparatus of claim 21, wherein the at least one hydrophilic polymer coating comprises polyethylene glycol (PEG).

27. The flow cell apparatus of claim 21, wherein one or more surfaces further comprise a second hydrophilic polymer coating.

28. The flow cell apparatus of claim 21, wherein the at least one hydrophilic polymer coating comprises a branched hydrophilic polymer.

29. The flow cell apparatus of claim 28, wherein the branched hydrophilic polymer comprises at least eight branches.

30. The flow cell apparatus of claim 23, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule, the single-stranded multimeric nucleic acid molecule comprising a repeating sequence of regularly occurring monomeric units.

31. The flow cell apparatus of claim 30, wherein the length of the single-stranded multimer nucleic acid molecule is at least 10 kilobases.

32. The flow cell apparatus of claim 30, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules further comprises a double-stranded monomer copy of the regularly occurring monomer unit.

33. The flow cell apparatus of claim 22, wherein the plurality of oligonucleotide molecules are present at various locations on the one or more surfaces with a substantially uniform surface density.

34. The flow cell apparatus of claim 22, wherein the plurality of oligonucleotide molecules are arranged at a rate of at least 100,000 molecules / μm. 2 A local surface density exists at a first location on the one or more surfaces, and a second local surface density exists at a second location on the one or more surfaces.

35. The flow cell apparatus of claim 10, wherein the coating comprises: (a) A first layer comprising a monolayer of polymer molecules tethered to the surface of one or more substrates; (b) a second layer comprising a second monolayer of polymer molecules tethered to the polymer molecules of the first layer; and (c) A third layer comprising a third monolayer polymer molecule tethered to the polymer molecule of the second layer, wherein at least one of the first layer, the second layer, or the third layer comprises a branched polymer molecule.

36. The flow cell apparatus of claim 35, wherein the third layer further comprises oligonucleotides tethered to the polymer molecules of the third layer.

37. The flow cell apparatus of claim 36, wherein the oligonucleotides tethered to the polymer molecules of the third layer are distributed throughout the third layer at multiple depths.

38. The flow cell apparatus of claim 17, wherein the coating further comprises: (a) A fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer, and (b) A fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer.

39. The flow cell apparatus of claim 38, wherein the polymer molecules of the fifth layer further comprise oligonucleotides tethered to the polymer molecules of the fifth layer.

40. The flow cell apparatus of claim 39, wherein the oligonucleotides tethered to the polymer molecules of the fifth layer are distributed throughout the fifth layer at multiple depths.

41. The flow cell apparatus of claim 21, wherein the at least one hydrophilic polymer coating comprises: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran.

42. The flow cell apparatus of claim 23, wherein when the plurality of clone-amplified sample nucleic acid molecules or their complementary sequences are labeled with cyanine dye-3, the images of the one or more surfaces show a fluorescence intensity of the cyanine dye-3-labeled sample nucleic acid molecules or their complementary sequences compared with the background of nonspecific cyanine dye-3 adsorption (B). 间隙 The ratio of fluorescence intensity of the two elements is at least 3:

1.

43. The flow cell apparatus of claim 42, wherein the fluorescence intensity of the sample nucleic acid molecules or their complementary sequences labeled with cyanine dye-3 exhibited by the image of one or more surfaces is combined with the background of nonspecific cyanine dye-3 adsorption and nonspecific amplification (B) 间隙 +B 内质 The ratio of fluorescence intensity of the two elements is at least 3:

1.

44. The flow cell apparatus of claim 23, wherein when the plurality of clone-amplified sample nucleic acid molecules or their complementary sequences are labeled with cyanine dye-3, the fluorescence intensity of the cyanine dye-3-labeled plurality of clone-amplified sample nucleic acid molecules or their complementary sequences as shown in the images of the one or more surfaces is compared with the background of nonspecific dye adsorption (B). 间隙 The ratio of fluorescence intensity of the two elements is at least 5:

1.

45. The flow cell apparatus of claim 44, wherein the fluorescence intensity of the sample nucleic acid molecules or their complementary sequences labeled with cyanine dye-3 exhibited by the images of said one or more surfaces is combined with the background of nonspecific cyanine dye-3 adsorption and nonspecific amplification (B) 间隙 +B 内质 The ratio of fluorescence intensity of the two elements is at least 5:

1.

46. ​​The flow cell apparatus of claim 23, wherein when the nucleic acid molecules or their complementary sequences of the plurality of cloned amplified samples are labeled with cyanine dye-3, and when the fluorescence images of the one or more surfaces are acquired under non-signal saturation conditions using an inverted microscope equipped with a 20× objective lens, NA=0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for cyanine dye-3 emission, and a camera, the fluorescence images exhibit a contrast-to-noise ratio (CNR) of at least 20.

47. The flow cell apparatus of claim 22, wherein the plurality of oligonucleotide molecules are in a flow rate of at least 1,000 molecules / m 2 The surface density exists.

48. The flow cell apparatus of claim 10, wherein the first reagent is configured to wet the one or more surfaces of the one or more channels.

49. The flow cell apparatus of claim 19, wherein the second reagent is configured to rewet the one or more surfaces of the one or more channels after at least a portion of moisture or liquid has been removed from the one or more surfaces of the one or more channels.

50. The flow cell apparatus of claim 1, wherein the air gap comprises air.

51. The flow cell apparatus of claim 1, wherein the air gap comprises dry air.

52. The flow cell apparatus of claim 1, wherein the air gap comprises one or more inert gases.

53. The flow cell apparatus according to claim 1, wherein the air gap comprises one or more active gases.

54. The flow cell apparatus of claim 5, wherein the first reagent or the second reagent comprises a liquid.

55. The flow cell apparatus according to claim 5, wherein the first reagent or the second reagent does not contain air bubbles larger than a predetermined size.

56. The flow cell apparatus of claim 17, wherein the coating comprises a liquid repellent coating.

57. The flow cell apparatus of claim 17, wherein the coating comprises a superhydrophobic coating.

58. The flow cell apparatus of claim 17, wherein the coating comprises a smooth liquid injection porous surface (SLIPS).

59. The flow cell apparatus of claim 17, wherein the coating comprises a smooth, superhydrophobic, covalently attached liquid (SOCAL) coating.

60. The flow cell apparatus of claim 17, wherein the coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates.

61. The flow cell apparatus of claim 17, wherein the coating is formed by impregnating a lubricant into one or more porous surfaces.

62. The flow cell apparatus of claim 61, wherein the lubricant comprises a surface energy of less than about 20 mJ / m 2 The liquid.

63. The flow cell apparatus of claim 61, wherein the lubricant comprises silicone oil.

64. The flow cell apparatus of claim 17, wherein the surface energy of the coating is less than about 20 mJ / m2.

65. The flow cell apparatus of claim 17, wherein the coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers.

66. The flow cell apparatus of claim 65, wherein the one or more brine monomers comprise dimethyldimethoxysilane.

67. The flow pool apparatus of claim 1, wherein the open landing area is fluidly connected to the one or more channels.

68. The flow pool apparatus of claim 1, wherein the open landing area is fluidly connected to one of the one or more channels.

69. The flow pool apparatus of claim 1, wherein the open landing area is fluidly connected to two or more of the one or more channels.

70. The flow pool apparatus of claim 1, wherein the open landing area is located on the bottom substrate of the one or more substrates.

71. The flow cell apparatus of claim 1, wherein the inlet comprises a hole in the top substrate of the one or more substrates.

72. The flow pool apparatus of claim 71, wherein the hole in the top substrate is positioned above at least a portion of the open landing area.

73. The flow cell apparatus of claim 72, wherein the flow cell apparatus is configured to allow a dispenser to openly dispense one or more reagents through the orifice to the open landing area.

74. The flow cell apparatus of claim 73, wherein the dispenser is configured to dispense the one or more reagents openly from the tip of the dispenser into the open landing area.

75. The flow cell apparatus of claim 74, wherein the dispenser is configured to dispense the one or more reagents openly from the tip of the dispenser into the open landing area without the need for conduit between the dispenser and the open landing area.

76. The flow pool apparatus of claim 75, wherein at least a portion of the tip of the dispenser contacts the open landing area.

77. The flow pool apparatus of claim 75, wherein the tip of the dispenser does not contact the open landing area.

78. The flow cell apparatus of claim 1, further comprising a clean outlet in one or more of the substrates.

79. The flow cell apparatus of claim 78, wherein the cleaning outlet is fluidly connected to the inlet.

80. The flow pool apparatus of claim 79, wherein the clean outlet is fluidly connected to the open landing area.

81. The flow pool apparatus of claim 80, wherein the clean outlet is located below the open landing area.

82. The flow cell apparatus of claim 78, wherein the cleaning outlet is located in the top or bottom substrate of the one or more substrates.

83. The flow cell apparatus of claim 78, wherein the cleaning outlet comprises a side port on the one or more substrates, wherein the side port: (a) Extends at least along a direction perpendicular to or nearly perpendicular to the x-direction; (b) Extends at least along a direction perpendicular to or nearly perpendicular to the y-direction; (c) Extends at least along a direction perpendicular to or nearly perpendicular to the z-direction; (d) Extends at least along the direction inclined to the x-direction; (e) Extends at least along a direction inclined to the y-direction; or (f) Extends at least along a direction inclined to the z-direction.

84. The flow cell apparatus of claim 78, wherein the cleaning outlet is configured to be connected to a first pump or a second pump.

85. The flow cell apparatus of claim 1, wherein the one or more channels comprise one or more microfluidic channels.

86. The flow cell apparatus of claim 10, wherein one or more surfaces are coated with fluorescent beads, the fluorescent beads being chemically fixed to the one or more surfaces.

87. The flow cell apparatus of claim 86, wherein the fluorescent beads are covalently attached to the one or more surfaces.

88. The flow cell apparatus of claim 13, wherein the gap between the inner top surface and the inner bottom surface is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm or 40 μm.

89. The flow cell apparatus of claim 1, wherein the height of the one or more channels is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm or 40 μm.

90. The flow cell apparatus of claim 18, wherein the polynucleotides captured thereon are configured for imaging in a sequencing cycle.

91. The flow cell apparatus of claim 1, wherein the one or more substrates comprise a top substrate and a bottom substrate.

92. The flow cell apparatus of claim 91, wherein one or more channels are defined between the top substrate and the bottom substrate.

93. The flow cell apparatus of claim 91, wherein the one or more channels are at least partially defined in the top surface of the bottom substrate.

94. The flow cell apparatus of claim 91, wherein the one or more channels are at least partially defined in the bottom surface of the top substrate.

95. The flow cell apparatus of claim 91, wherein the one or more substrates further comprises an intermediate substrate.

96. The flow cell apparatus of claim 95, wherein one or more channels are at least partially defined in the intermediate substrate.

97. The flow cell apparatus of claim 1, wherein the one or more substrates comprise glass or plastic.

98. The flow cell apparatus of claim 1, wherein at least a portion of the carrier is transparent.

99. The flow cell apparatus of claim 1, wherein at least a portion of the one or more substrates is transparent.

100. The flow cell apparatus according to claim 1, wherein the carrier is solid.

101. The flow cell apparatus according to claim 1, wherein the one or more channels comprise 1, 2, 3, 4, 5, 6, 7 or 8 channels.

102. The flow cell apparatus of claim 1, wherein the one or more channels comprise 2, 4, 6, 8 or 10 channels.

103. The flow pool apparatus of claim 1, wherein the lane length of each of the one or more channels is less than about 70 mm, 75 mm, 80 mm or 90 mm.

104. The flow pool apparatus of claim 1, wherein the lane width of each of the one or more channels is less than about 10 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm or 3 mm.

105. The flow pool apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising a smooth coating.

106. The flow pool apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating comprising a liquid repellent coating.

107. The flow pool apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising a superhydrophobic coating.

108. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating comprising a smooth liquid injection porous surface (SLIPS).

109. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating comprising a smooth, superhydrophobic, covalently attached liquid (SOCAL) coating.

110. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising a liquid-like polymer brush surface covalently attached to the one or more substrates.

111. The flow pool apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating, the second surface coating comprising impregnating a lubricant in a porous surface to produce a second surface coating with a surface energy of less than about 20 mJ / m2.

112. The flow cell apparatus of claim 2, wherein at least a portion of the open landing area is covered with a second surface coating comprising acid-catalyzed graft polycondensation of one or more brine monomers.

113. The flow cell apparatus of claim 112, wherein the one or more brine monomers comprise dimethyldimethoxysilane.

114. The flow cell apparatus of claim 5, wherein the flow cell apparatus is configured to allow cleaning of at least a portion of the first reagent from at least a portion of the one or more channels during a DNA sequencing run.

115. The flow cell apparatus of claim 5, wherein the flow cell apparatus is configured to allow at least a portion of the first reagent to remain in the one or more channels.

116. The flow cell apparatus according to claim 5, wherein the first reagent and the second reagent are different.

117. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than 40% of the corresponding volume or length of each of the one or more channels.

118. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than half of the corresponding volume or length of each of the one or more channels.

119. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than 60% of the corresponding volume or length of each of the one or more channels.

120. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than 70% of the corresponding volume or length of each of the one or more channels.

121. The flow cell apparatus of claim 1, wherein at least a portion of the one or more channels occupies more than 80% of the corresponding volume or length of each of the one or more channels.

122. The flow cell apparatus of claim 5, wherein the clean outlet is configured to allow residual amounts of the first reagent on the open landing area to flow through the clean outlet.

123. The flow cell apparatus of claim 122, wherein the residual amount of the first reagent in the open landing area comprises the meniscus of the first reagent.

124. The flow cell apparatus of claim 91, further comprising one or more seals positioned on the one or more substrates.

125. The flow cell apparatus of claim 124, wherein a first portion of one or more channels includes a first z-position, and a second portion of the channel includes a second z-position different from the first z-position.

126. The flow cell apparatus of claim 125, wherein the first portion of the channel comprises one or more first imaging surfaces.

127. The flow cell apparatus of claim 125, wherein the second portion of the channel comprises one or more second imaging surfaces.

128. The flow cell apparatus of claim 125, wherein the top substrate or the bottom substrate comprises one or more substrate layers.

129. The flow cell apparatus of claim 125, wherein the top substrate includes a first thickness above the first portion of the channel and a second thickness around the second portion of the channel.

130. The flow cell apparatus of claim 129, wherein the second thickness is greater than the first thickness.

131. The flow cell apparatus of claim 129, wherein the second thickness is 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the first thickness.

132. The flow cell apparatus of claim 125, wherein the bottom substrate includes a third thickness above the first portion of the channel and a fourth thickness above the second portion of the channel.

133. The flow cell apparatus of claim 132, wherein the fourth thickness is greater than the third thickness.

134. The flow cell apparatus of claim 132, wherein the fourth thickness is 20%, 50%, 80%, 100%, 120%, 150%, or 200% greater than the third thickness.

135. The flow cell apparatus of claim 124, wherein the one or more seals comprise one or more mechanical seals.

136. The flow cell apparatus of claim 124, wherein the one or more seals comprise one or more gaskets.

137. The flow cell apparatus of claim 79, wherein the cleaning outlet is configured to remove fluid from the one or more channels.

138. The flow cell apparatus of claim 79, wherein the cleaning outlet is in sealed fluid connection with a pump or vacuum device.

139. The flow cell apparatus of claim 79, wherein the cleaning outlet is configured to direct fluid or gas to the one or more channels.

140. The flow cell apparatus of claim 91, wherein the bottom substrate comprises glass, plastic, or both.

141. The flow cell apparatus of claim 125, wherein the one or more seals comprise a first seal having a thickness along the z-direction equivalent to the thickness of the top substrate in the second portion.

142. The flow cell apparatus of claim 125, wherein the one or more seals include a second seal, the thickness of the second seal along the z-direction being comparable to the thickness of the bottom substrate in the second portion.

143. The flow cell apparatus of claim 142, wherein the thickness of the second seal along the z-direction is greater than the thickness of the bottom substrate in the first portion.

144. The flow cell apparatus of claim 124, wherein the flow cell apparatus further comprises a frame that covers at least a portion of the one or more substrates.

145. The flow cell apparatus of claim 144, wherein the frame is mechanically fixed to the one or more seals.

146. The flow cell apparatus of claim 144, wherein the frame comprises plastic.

147. The flow cell apparatus of claim 125, wherein the one or more seals interface with a manifold or connector to allow sealed fluid communication between the manifold or connector and the one or more channels.

148. The flow cell apparatus of claim 147, wherein the manifold or the connector comprises one or more fluid passages.

149. The flow cell apparatus of claim 148, wherein the one or more fluid passages are in fluid communication with the one or more channels.

150. The flow pool apparatus of claim 148, wherein one or more fluid passages are in fluid communication with the open landing area.

151. The flow cell apparatus of claim 148, wherein the manifold or the connector is configured to be in sealed fluid communication with the one or more channels by applying a pressure thereon that satisfies a predetermined threshold.

152. The flow cell apparatus of claim 151, wherein one or more fluid passages extend along the y-axis, and wherein the pressure is applied along the y-axis.

153. The flow cell apparatus of claim 151, wherein one or more fluid passages extend along the x-axis, and wherein pressure is applied along the x-axis.

154. The flow cell apparatus of claim 148, wherein the manifold or the connector includes a coupling interface that directly contacts an end of the flow cell apparatus.

155. The flow cell device of claim 148, wherein the manifold or the connector includes a mating interface that contacts an end of the flow cell device with the one or more seals therebetween.

156. The flow cell apparatus of claim 148, wherein the manifold or the connector includes a mating interface that contacts an end of the flow cell apparatus with an adhesive therebetween.

157. The flow cell apparatus of claim 148, wherein the manifold or the connector includes an open region located at the end of one or more of the fluid passages.

158. The flow pool apparatus of claim 152, wherein the open region is fluidly connected to the open landing region.

159. The flow cell device of claim 148, further comprising one or more reference features configured to position the flow cell device relative to the manifold or the connector, sample stage or sequencing system.

160. The flow cell apparatus of claim 159, wherein the one or more reference features comprise at least one alignment feature, the at least one alignment feature being positioned at a center point along the x-axis.

161. The flow cell apparatus of claim 159, wherein the one or more reference features comprise at least one alignment feature, the at least one alignment feature being positioned along the y-axis at or near an end of the one or more substrates.

162. The flow cell apparatus of claim 159, wherein the one or more reference features include a cavity extending through the one or more substrates and configured to be coupled to a pin.

163. The flow cell apparatus of claim 159, wherein the one or more reference features include a groove extending through the one or more substrates, the groove being configured to engage with a pin.

164. The flow cell apparatus of claim 159, wherein the manifold or the connector comprises a top portion or a bottom portion extending beyond the one or more substrates along the z-axis and covering at least a portion of the one or more substrates in the xy-plane.

165. The flow cell apparatus of claim 164, wherein the top portion or the bottom portion is located at the first portion, the second portion, or both of the one or more channels.

166. The flow cell apparatus of claim 164, wherein the top portion or the bottom portion includes one or more alignment features configured to align the top portion or the bottom portion with the flow cell apparatus.

167. The flow cell apparatus of claim 164, wherein the top portion or the bottom portion includes one or more alignment features configured to align the top portion or the bottom portion with the flow cell apparatus along the z-axis or along the y-axis.

168. The flow cell apparatus of claim 148, further comprising one or more tubes, the one or more tubes interfacing with the manifold or the connector and the flow cell apparatus.

169. The flow cell apparatus of claim 168, wherein each of the one or more tubes comprises a wall surrounding a lumen.

170. The flow cell apparatus of claim 169, wherein the lumen is in fluid communication with one or more channels of the flow cell apparatus and one or more fluid passages of the manifold or the connector.

171. The flow cell apparatus of claim 170, wherein at least a portion of the one or more tubes is embedded in the one or more substrates.

172. The flow cell apparatus of claim 171, wherein each of the one or more pipes is connected to the manifold or the connector, thereby enabling fluid communication therebetween.

173. The flow cell apparatus of claim 125, wherein the one or more seals comprise a protective sleeve seal that covers at least a portion of the flow cell apparatus in the xy plane and covers one end of the flow cell apparatus in the xz plane.

174. The flow cell apparatus of claim 125, wherein the one or more seals comprise a flexible material that deforms under pressure satisfying a predetermined threshold.

175. The flow cell apparatus of claim 125, wherein the one or more seals comprise an L-shaped seal extending along the z-axis and the y-axis.

176. The flow cell apparatus of claim 175, wherein the L-shaped seal extends along the y-axis and into a corresponding channel in one or more of the channels.

177. The flow cell device of claim 176, wherein pressure or force is applied to the L-shaped seal along the y-axis to enable a sealed fluid communication between the flow cell device and the manifold.

178. The flow cell device of claim 148, wherein one or more seals are configured to interface with the manifold or connector, thereby allowing sealed fluid communication between the flow cell device and the manifold.

179. The flow cell apparatus of claim 125, wherein the one or more seals comprise membrane seals that cover at least a portion of the flow cell apparatus and at least a portion of the manifold or the connector, thereby sealing the fluid communication therebetween.

180. The flow cell apparatus of claim 179, wherein the membrane seal comprises a flat washer placed on top of the top surface of the top substrate, a flat washer placed below the bottom surface of the bottom substrate, or both.

181. The flow cell apparatus of claim 179, wherein the membrane seal extends in the xy plane.

182. The flow cell apparatus of claim 148, wherein the manifold or connector includes a finger-shaped cutout region located between two channels of the one or more channels of the flow cell apparatus.

183. The flow cell apparatus of claim 182, wherein the manifold or the connector comprises a seal placed in the finger-cut region and configured to seal the fluid communication between the two channels.

184. The flow cell apparatus of claim 148, wherein the manifold or the connector includes a fluid passage having an outlet that exits the manifold in a plane orthogonal to the y-axis, the x-axis, or the z-axis.

185. The flow cell apparatus of claim 125, wherein the top substrate or the bottom substrate comprises one or more inclined ends.

186. The flow cell apparatus of claim 185, wherein the tip of one of the one or more inclined ends presses against the one or more seals.

187. The flow cell apparatus of claim 186, wherein each of the one or more inclined ends interfaces with an inclined manifold or connector.

188. The flow cell apparatus of claim 187, wherein the one or more inclined ends comprise a first acute ramp angle relative to the y-axis.

189. The flow cell apparatus of claim 188, wherein the inclined manifold or connector includes a second acute ramp angle relative to the y-axis.

190. The flow cell apparatus of claim 189, wherein the first acute ramp angle is different from the second acute ramp angle.

191. The flow cell apparatus according to claim 190, wherein the first acute ramp angle is the same as the second acute ramp angle.

192. The flow cell apparatus of claim 191, wherein the inclined manifold or connector includes a ramp complementary to the inclined end of the flow cell apparatus.

193. The flow cell apparatus of claim 192, wherein the one or more seals comprise diagonal washers having fluid passages extending in the yz plane.

194. The flow cell apparatus of claim 193, wherein the diagonal washer, the manifold, or the connector interfaces with an end of the top substrate and a top surface of the bottom substrate.

195. The flow cell apparatus of claim 193, wherein the diagonal washer, the manifold, or the connector interfaces with an end of the bottom substrate and the top inner surface of the top substrate.

196. The flow cell apparatus of claim 193, wherein when the force or pressure includes a y-axis component satisfying a first threshold and a z-axis component satisfying a second threshold, the diagonal gasket manifold or the connector allows sealed fluid communication from the fluid passage to the one or more channels.

197. The flow cell apparatus of claim 148, wherein the top substrate and the bottom substrate are offset from each other at least laterally along the y-axis.

198. The flow cell apparatus of claim 148, wherein at least a portion of the manifold or the connector is fixedly attached to the bottom inner surface of the bottom substrate.

199. The flow cell apparatus of claim 148, further comprising an intermediary layer configured to define the one or more channels between the top substrate and the bottom substrate.

200. The flow cell apparatus of claim 148, wherein the top substrate and the bottom substrate are not directly and fixedly attached to each other.

201. The flow cell apparatus of claim 148, wherein at least a portion of the manifold or the connector is fixedly attached to the top inner surface of the top substrate.

202. The flow cell apparatus of claim 196, wherein the fluid passage of the diagonal washer, the manifold, or the connector extends at least along the y-axis.

203. The flow cell apparatus of claim 148, wherein the manifold or the connector further comprises an open well leading to a second open landing area, and wherein the second open landing area is configured to receive reagent from a dispensing tip.

204. The flow pool apparatus of claim 203, wherein the open well of the manifold or the connector is in fluid communication with the one or more channels.

205. The flow pool apparatus of claim 204, wherein the second open landing area of ​​the manifold or the connector is in fluid communication with the inlet of the one or more channels.

206. The flow cell apparatus of claim 125, wherein the one or more seals comprise a thermoplastic connector and a thermoplastic seal mounted on the thermoplastic connector.

207. The flow cell apparatus of claim 206, wherein the thermoplastic seal is deformable under pressure changes, temperature changes, or both.

208. The flow cell apparatus of claim 207, wherein the thermoplastic seal comprises one or more materials, the one or more materials being different from the one or more materials of the thermoplastic connector.

209. The flow cell apparatus of claim 125, wherein the one or more seals comprise a first connector having a top portion that is slidable on a top surface of the top substrate.

210. The flow cell apparatus of claim 209, wherein the one or more seals include a second connector having a bottom portion that is slidable on the bottom surface of the bottom substrate.

211. The flow cell apparatus of claim 210, wherein the top portion is connected to a first side portion of the first connector, the first side portion being configured to interface with an end of the flow cell apparatus in the xz plane.

212. The flow cell device of claim 210, wherein the bottom portion is connected to a second side portion of the second connector, the second side portion being configured to interface with an end of the flow cell device in the xz plane.

213. The flow cell device of claim 212, wherein the pressure or force satisfying a predetermined threshold on the first side portion and the second side portion is configured to cause the first connector and the second connector to slide relative to the flow cell device under deformation, thereby enabling the one or more channels to be in sealed communication with the fluid passage defined between the top connector and the bottom connector.

214. The flow cell apparatus of claim 125, wherein the inlet comprises a port that opens at the bottom surface of the bottom substrate.

215. The flow cell apparatus of claim 214, wherein the port is in fluid communication with one or more channels of the connector and the fluid passage.

216. The flow cell apparatus of claim 125, wherein the one or more seals comprise a semi-rigid or deformable material that deforms under pressure or force.

217. The flow cell apparatus of claim 216, wherein the semi-rigid or deformable material is configured to recover its shape prior to deformation when the pressure or force is removed.

218. The flow cell apparatus of claim 125, wherein the one or more seals comprise a gasket, a second connector, a second manifold or a component thereof, or a combination thereof.

219. The flow cell apparatus of claim 218, further comprising a force application mechanism controlled by computer-readable instructions executable on a computer processor.

220. The flow cell device of claim 219, wherein the second manifold, the second connector, or the one or more seals are connected to the force application mechanism, thereby allowing connection to or disconnection from the flow cell device.

221. A flow cell system comprising: (a) The flow cell apparatus according to any one of claims 1 to 220; (b) Fluid control device.

222. The flow cell system of claim 221, wherein the fluid control device comprises a first pump, a second pump, or both.

223. The flow cell system of claim 221, wherein the fluid control device comprises: (a) a third pump, said third pump being connected to the outlet of the flow tank device; and (b) A dispenser configured to openly dispense the one or more reagents into the inlet of the flow cell device.

224. The flow cell system of claim 221, wherein the fluid control device comprises: (a) A fourth pump, which is fluidly connected to the clean outlet of the flow tank device; (b) a fifth pump, wherein the fourth pump or the fifth pump is fluidly connected to the outlet of the flow tank apparatus; and (c) A dispenser configured to openly dispense the one or more reagents to the inlet of the flow cell device.

225. The flow pool system of any one of claims 221 to 224, wherein the first pump or the second pump is configured to introduce the air gap through the inlet and cause the air gap to flow at least partially through the one or more channels.

226. The flow cell system according to any one of claims 221 to 225, further comprising a third manifold or connector having the fluid passage extending in the yz plane.

227. The flow cell system according to any one of claims 221 to 226, wherein the first pump is configured to clean the open landing area by displacing the residual amount of the first reagent away from the open landing area to flow through the clean outlet.

228. A method for preparing a flow cell for a DNA sequencing reaction, the method comprising: (a) Providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet includes an open landing region for receiving one or more reagents, and (ii) one or more channels disposed between the inlet and the outlet to perform the sequencing reaction; (b) Dispensing a first reagent of the one or more reagents into the open landing area such that at least a portion of the first reagent flows from the open landing area into the one or more channels; (c) Introducing gas into the one or more channels; (d) The second reagent of the one or more reagents is openly dispensed into the open landing area so that at least a portion of the second reagent flows from the open landing area into the one or more channels, thereby removing residual amounts of the first reagent from the one or more channels.

229. A method for preparing a flow cell for a DNA sequencing reaction, the method comprising: (a) Providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet includes an open landing region for receiving one or more reagents, and (ii) one or more channels disposed between the inlet and the outlet to perform the sequencing reaction; (b) Dispensing a first reagent of the one or more reagents into the open landing area such that at least a portion of the first reagent flows from the open landing area into the one or more channels; The open landing area at least partially comprises a surface coating to facilitate the removal of residual amounts of the first reagent from the open landing area; as well as (c) Dispensing a second reagent from the one or more reagents into the open landing area such that at least a portion of the second reagent flows from the open landing area into the one or more channels.

230. A method for sequencing using a flow cell apparatus, the method comprising: (a) Providing the flow cell, the flow cell comprising (i) an inlet and an outlet, wherein the inlet includes an open landing region for receiving one or more reagents, and (ii) one or more channels disposed between the inlet and the outlet to perform the sequencing reaction; (b) Dispensing a first reagent of the one or more reagents into the open landing area such that at least a portion of the first reagent flows from the open landing area into the one or more channels; (c) The residual amount of the first reagent is removed from the open landing area by allowing the residual amount of the first reagent to flow through the clean outlet of the flow cell device; as well as (d) Dispensing a second reagent from the one or more reagents into the open landing area, such that at least a portion of the second reagent flows from the open landing area into the one or more channels.

231. A method for manufacturing a flow cell apparatus, the method comprising: Obtain one or more substrates; One or more channels are formed in the one or more substrates, wherein the one or more channels are configured to allow fluid or an air gap between the fluid and another fluid to flow through the one or more channels; An inlet is formed, the inlet comprising a hole in one of the one or more substrates and an open landing area, wherein the inlet is fluidly connected to the one or more channels; An outlet is formed, which is fluidly connected to the one or more channels; At least a portion of the surface of the one or more channels is coated with a first coating, wherein the surface is configured to be dried and re-wetted during DNA sequencing runs; as well as One of the one or more substrates is fixedly connected together.

232. A method for manufacturing a flow cell apparatus, the method comprising: Obtain one or more substrates; One or more channels are generated in the one or more substrates; An inlet is formed, the inlet comprising a hole in one of the one or more substrates and an open landing area, wherein the inlet is fluidly connected to the one or more channels; At least a portion of the surface of the one or more channels is coated with a first coating; Cover at least a portion of the open landing area with a second coating; as well as One of the one or more substrates is fixedly connected together.

233. A method for manufacturing a flow cell apparatus, the method comprising: Obtain one or more substrates; An inlet is formed, the inlet comprising a hole in one of the one or more substrates and an open landing area; One or more channels are generated in the one or more substrates; An outlet is formed in one or more substrates, wherein the inlet and the outlet are fluidly connected to the one or more channels; A cleaning outlet is formed in one or more substrates, wherein the cleaning outlet is fluidly connected to the inlet, and wherein the cleaning outlet is closer to the inlet than to the outlet. as well as One of the one or more substrates is fixedly connected together.

234. The method of any one of claims 231 to 233, wherein the one or more channels are configured to allow air gap flow through the one or more channels between allowing the first reagent and the second reagent to flow through the one or more channels.

235. The method of any one of claims 231 to 234, wherein the one or more channels are configured to allow air gaps to flow through the one or more channels during DNA sequencing runs.

236. The method of any one of claims 231 to 235, wherein the one or more channels are configured to allow the air gap to flow from the inlet through the one or more channels.

237. The method of any one of claims 231 to 236, wherein the one or more channels are configured to allow air gap flow through the one or more channels to facilitate reduced contamination of the second reagent by the first reagent during DNA sequencing runs.

238. The method of any one of claims 231 to 237, wherein the one or more channels are configured to allow air gap flow through the one or more channels to reduce the minimum amount of the first reagent, the second reagent, or the washing reagent required for a DNA sequencing run.

239. The method according to any one of claims 231 to 238, wherein one of the one or more channels comprises one or more surfaces.

240. The method of claim 239, wherein the one or more surfaces comprise an inner surface.

241. The method of claim 239, wherein the one or more surfaces comprise an outer surface.

242. The method of claim 239, wherein the one or more surfaces comprise an inner top surface, an inner bottom surface, or both.

243. The method of claim 239, wherein the one or more surfaces comprise an outer top surface, an outer bottom surface, or both.

244. The method of claim 239, wherein the one or more surfaces comprise planar surfaces.

245. The method of claim 239, wherein the one or more surfaces are passivated.

246. The method of claim 239, wherein the one or more surfaces are passivated with a coating that immobilizes the surface to capture primers, nucleic acid template molecules, or both, to capture polynucleotides.

247. The method of claim 239, wherein the one or more surfaces comprise polynucleotides captured thereon.

248. The method of any one of claims 231 to 247, wherein the air gap is configured to dry at least a portion of the one or more surfaces of the one or more channels.

249. The method according to any one of claims 239 to 248, wherein the air gap does not impair the chemical function of the one or more surfaces.

250. The method according to any one of claims 231 to 249, wherein the coating on the one or more surfaces comprises at least one hydrophilic polymer coating.

251. The method according to any one of claims 231 to 250, wherein the coating on one or more surfaces comprises a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating.

252. The method according to any one of claims 239 to 251, wherein the one or more surfaces comprise at least one discrete region, the at least one discrete region comprising a plurality of cloned amplified sample nucleic acid molecules, the plurality of cloned amplified sample nucleic acid molecules having been annealed to a plurality of attached oligonucleotide molecules.

253. The method of claim 251, wherein the water contact angle of the at least one hydrophilic polymer coating does not exceed about 50 degrees.

254. The method of claim 252, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a tandem polymer, the tandem polymer being annealed to at least one of the plurality of attached oligonucleotide molecules.

255. The method of claim 251, wherein the at least one hydrophilic polymer coating comprises PEG.

256. The method of claim 251, wherein the one or more surfaces further comprise a second hydrophilic polymer coating.

257. The method of claim 251, wherein the at least one hydrophilic polymer coating comprises a branched hydrophilic polymer.

258. The method of claim 257, wherein the branched hydrophilic polymer comprises at least eight branches.

259. The method of claim 252, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule, the single-stranded multimeric nucleic acid molecule comprising a repeating sequence of regularly occurring monomeric units.

260. The method of claim 259, wherein the length of the single-stranded multimeric nucleic acid molecule is at least 10 kilobases.

261. The method of claim 259, wherein at least one of the plurality of cloned amplified sample nucleic acid molecules further comprises a double-stranded monomer copy of the regularly occurring monomer unit.

262. The method of claim 251, wherein the plurality of oligonucleotide molecules are present at various locations on the one or more surfaces with a substantially uniform surface density.

263. The method of claim 251, wherein the plurality of oligonucleotide molecules are present in a first region on the one or more surfaces at a local surface density of at least 100,000 molecules / μm², and in a second region on the one or more surfaces at a second local surface density.

264. The method of claim 246, wherein the coating comprises: (a) A first layer comprising a monolayer of polymer molecules tethered to one or more surfaces of the substrate; (b) a second layer comprising a second monolayer of polymer molecules tethered to the polymer molecules of the first layer; and (c) A third layer comprising a third monolayer polymer molecule tethered to the polymer molecule of the second layer, wherein at least one of the first layer, the second layer, or the third layer comprises a branched polymer molecule.

265. The method of claim 264, wherein the third layer further comprises an oligonucleotide tethered to the polymer molecule of the third layer.

266. The method of claim 265, wherein the oligonucleotides tethered to the polymer molecules of the third layer are distributed throughout the third layer at multiple depths.

267. The method of claim 264, wherein the coating further comprises: (a) A fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer, and (b) A fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer.

268. The method of claim 267, wherein the polymer molecules of the fifth layer further comprise oligonucleotides tethered to the polymer molecules of the fifth layer.

269. The method of claim 268, wherein the oligonucleotides tethered to the polymer molecules of the fifth layer are distributed throughout the fifth layer at multiple depths.

270. The method of claim 250, wherein the at least one hydrophilic polymer coating comprises: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran.

271. The method of claim 252, wherein when the cloned amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine dye-3, the images of the one or more surfaces show a fluorescence intensity of the cloned amplified, cyanine dye-3 labeled sample nucleic acid molecule or its complementary sequence and a background of nonspecific cyanine dye-3 adsorption (B). 间隙 The ratio of fluorescence intensity of the two elements is at least 3:

1.

272. The method of claim 271, wherein the fluorescence intensity of the image of said one or more surfaces exhibiting a combination of the fluorescence intensity of the clonal amplified, anthocyanin-3 labeled sample nucleic acid molecule or its complementary sequence with the background of nonspecific anthocyanin-3 dye adsorption and nonspecific amplification (B) 间隙 +B 内质 The ratio of fluorescence intensity of the two elements is at least 3:

1.

273. The method of claim 252, wherein when the cloned amplified sample nucleic acid molecule or its complementary sequence is labeled with cyanine dye-3, the ratio of the fluorescence intensity of the cloned amplified, cyanine dye-3 labeled sample nucleic acid molecule or its complementary sequence shown in the images of the one or more surfaces to the fluorescence intensity of the nonspecific dye adsorption background (B gap) is at least 5:

1.

274. The method of claim 273, wherein the fluorescence intensity of the image of said one or more surfaces exhibiting a combination of the fluorescence intensity of the clonal amplified, anthocyanin-3 labeled sample nucleic acid molecule or its complementary sequence with the background of nonspecific anthocyanin-3 dye adsorption and nonspecific amplification (B) 间隙 +B 内质 The ratio of fluorescence intensity of the two elements is at least 5:

1.

275. The method of claim 252, wherein, when the sample nucleic acid molecule or its complementary sequence of the cloned amplified sample is labeled with cyanine dye-3, and when the fluorescence images of the one or more surfaces are acquired under non-signal saturation conditions using an inverted microscope equipped with a 20× objective lens, NA=0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for cyanine dye-3 emission, and a camera, the fluorescence images exhibit a contrast-to-noise ratio (CNR) of at least 20.

276. The method of claim 251, wherein the plurality of oligonucleotide molecules are arranged in an order of at least 1,000 molecules / m 2 The surface density exists.

277. The method according to any one of claims 231 to 276, wherein the first reagent is configured to wet the one or more surfaces of the one or more channels.

278. The method according to any one of claims 231 to 277, wherein the second reagent is configured to rewet the one or more surfaces of the one or more channels after the one or more surfaces have been at least partially dried through the air gap.

279. The method according to any one of claims 231 to 278, wherein the flow cell system includes the flow cell apparatus, wherein the flow cell system further includes a fluid control device, the fluid control device comprising: (a) a first pump, the first pump being connected to the outlet; and (b) A dispenser configured to openly dispense one or more reagents into the inlet.

280. The method of any one of claims 231 to 279, wherein the first pump or the second pump is configured to introduce the air gap through the inlet and cause the air gap to flow at least partially through the one or more channels.

281. The method according to any one of claims 231 to 280, wherein the air gap comprises air.

282. The method according to any one of claims 231 to 280, wherein the air gap comprises dry air.

283. The method according to any one of claims 231 to 280, wherein the air gap comprises one or more inert gases.

284. The method according to any one of claims 231 to 280, wherein the air gap comprises one or more active gases.

285. The method according to any one of claims 231 to 284, wherein the first reagent or the second reagent comprises a liquid.

286. The method according to any one of claims 231 to 285, wherein the first reagent or the second reagent lacks bubbles larger than a predetermined size.

287. The method according to any one of claims 246 to 286, wherein the coating comprises a liquid repellent coating.

288. The method according to any one of claims 246 to 286, wherein the coating comprises a superhydrophobic coating.

289. The method according to any one of claims 246 to 286, wherein the coating comprises a smooth liquid-filled porous surface (SLIPS).

290. The method of any one of claims 246 to 286, wherein the coating comprises a smooth superhydrophobic covalently attached liquid (SOCAL) coating.

291. The method of any one of claims 246 to 286, wherein the coating comprises a liquid-like polymer brush surface covalently attached to the one or more substrates.

292. The method according to any one of claims 246 to 286, wherein the coating is formed by impregnating a lubricant into one or more porous surfaces.

293. The method of claim 292, wherein the lubricant comprises a surface energy of less than about 20 mJ / m 2 The liquid.

294. The method of claim 292, wherein the lubricant comprises silicone oil.

295. The method according to any one of claims 246 to 286, wherein the surface energy of the coating is less than about 20 mJ / m 2 .

296. The method according to any one of claims 246 to 286, wherein the coating is formed by acid-catalyzed graft polycondensation of one or more brine monomers.

297. The method of claim 296, wherein the one or more brine monomers comprise dimethyldimethoxysilane.

298. The method according to any one of claims 231 to 297, wherein the open landing area is fluidly connected to the one or more channels.

299. The method according to any one of claims 231 to 297, wherein the open landing area is fluidly connected to one of the one or more channels.

300. The method of any one of claims 231 to 297, wherein the open landing area is located on the bottom substrate of the one or more substrates.

301. The method according to any one of claims 231 to 300, wherein the inlet comprises a hole in the top substrate of the one or more substrates.

302. The method of claim 301, wherein the hole in the top substrate is positioned above at least a portion of the open landing area.

303. The method according to any one of claims 279 to 302, wherein the dispenser is configured to openly dispense the one or more reagents through the orifice to the open landing area.

304. The method according to any one of claims 279 to 302, wherein the dispenser is configured to dispense the one or more reagents openly from the tip of the dispenser into the open landing area.

305. The method according to any one of claims 279 to 302, wherein the dispenser is configured to dispense the one or more reagents openly from the tip of the dispenser into the open landing area without the need for a conduit between the dispenser and the open landing area.

306. The method of claim 305, wherein at least a portion of the tip of the dispenser contacts the open landing area.

307. The method of claim 305, wherein the tip of the dispenser does not contact the open landing area.

308. The method according to any one of claims 231 to 307, wherein the flow cell device further comprises a cleaning outlet in one or more substrates.

309. The method of claim 308, wherein the cleaning outlet is fluidly connected to the inlet.

310. The method of claim 308, wherein the clean outlet is fluidly connected to the open landing area.

311. The method of claim 308, wherein the cleaning outlet is located in the top or bottom substrate of the one or more substrates.

312. The method of claim 308, wherein the cleaning outlet comprises a side port on the one or more substrates, wherein the side port: (a) Extends at least along a direction perpendicular to or nearly perpendicular to the x-direction; (b) Extends at least along a direction perpendicular to or nearly perpendicular to the y-direction; (c) Extends at least along a direction perpendicular to or nearly perpendicular to the z-direction; (d) Extends at least along the direction inclined to the x-direction; (e) Extends at least along a direction inclined to the y-direction; or (f) Extends at least along a direction inclined to the z-direction.

313. The method of claim 308, wherein the cleaning outlet is configured to be connected to the first pump or the second pump.

314. The method according to any one of claims 231 to 313, wherein the one or more channels comprise microfluidic channels.

315. The method according to any one of claims 231 to 314, wherein the one or more surfaces are coated with fluorescent beads, the fluorescent beads being chemically fixed to the one or more surfaces.

316. The method of claim 315, wherein the fluorescent beads are covalently attached to the one or more surfaces.

317. The method according to any one of claims 231 to 316, wherein the gap between the inner top surface and the inner bottom surface is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm or 40 μm.

318. The method according to any one of claims 231 to 316, wherein the height of the one or more channels is about 150 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm or 40 μm.

319. The method of any one of claims 247 to 318, wherein the polynucleotides captured thereon are configured to be imaged in a sequencing cycle.

320. The method according to any one of claims 231 to 319, wherein the one or more substrates comprise a top substrate and a bottom substrate.

321. The method of claim 320, wherein the one or more channels are defined between the top substrate and the bottom substrate.

322. The method of claim 320, wherein the one or more channels are at least partially defined in the top surface of the bottom substrate.

323. The method of claim 320, wherein the one or more channels are at least partially defined in the bottom surface of the top substrate.

324. The method of claim 320, wherein the one or more substrates further comprises an intermediate substrate.

325. The method of claim 324, wherein the one or more channels are at least partially defined in the intermediate substrate.

326. The method according to any one of claims 231 to 325, wherein the one or more substrates comprise glass or plastic.

327. The method according to any one of claims 231 to 326, wherein at least a portion of the carrier is transparent.

328. The method according to any one of claims 231 to 327, wherein at least a portion of the one or more substrates is transparent.

329. The method according to any one of claims 231 to 328, wherein the carrier is solid.

330. The method according to any one of claims 231 to 329, wherein the one or more channels comprise 1, 2, 3, 4, 5, 6, 7 or 8 channels.

331. The method according to any one of claims 231 to 330, wherein the one or more channels comprise 2, 4, 6, 8 or 10 channels.

332. The method according to any one of claims 231 to 331, wherein the lane length of each of the one or more channels is less than about 70 mm, 75 mm, 80 mm or 90 mm.

333. The method according to any one of claims 231 to 331, wherein the lane width of each of the one or more channels is less than about 10 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm or 3 mm.

334. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating, the second coating comprising a smooth coating.

335. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating comprising a liquid repellent coating.

336. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating, the second coating comprising a superhydrophobic coating.

337. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating comprising a smooth liquid-filled porous surface (SLIPS).

338. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating comprising a smooth, superhydrophobic, covalently attached liquid (SOCAL) coating.

339. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating comprising a liquid-like polymer brush surface covalently attached to the one or more substrates.

340. The method of any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating comprising impregnating a lubricant in a porous surface to produce a surface energy of less than about 20 mJ / m².

341. The method according to any one of claims 231 to 333, wherein at least a portion of the open landing area comprises a second coating comprising an impregnated acid-catalyzed graft polycondensation of one or more brine monomers.

342. The method of claim 341, wherein the one or more brine monomers comprise dimethyldimethoxysilane.

343. The method according to any one of claims 231 to 342, wherein the process of using the flow cell device comprises removing at least a portion of the first reagent from at least a portion of the one or more channels during a DNA sequencing run.

344. The method of claim 343, wherein at least a portion of the first reagent is retained in the one or more channels during the DNA sequencing run.

345. The method according to any one of claims 231 to 344, wherein the first reagent and the second reagent are different.

346. The method according to any one of claims 231 to 345, wherein at least a portion of the one or more channels accounts for more than 40% of the corresponding volume or length of each of the one or more channels.

347. The method according to any one of claims 231 to 345, wherein at least a portion of the one or more channels comprises more than half of the corresponding volume or length of each of the one or more channels.

348. The method according to any one of claims 231 to 345, wherein at least a portion of the one or more channels accounts for more than 60% of the corresponding volume or length of each of the one or more channels.

349. The method according to any one of claims 231 to 345, wherein at least a portion of the one or more channels accounts for more than 70% of the corresponding volume or length of each of the one or more channels.

350. The method according to any one of claims 231 to 345, wherein at least a portion of the one or more channels accounts for more than 80% of the corresponding volume or length of each of the one or more channels.

351. The method according to any one of claims 231 to 350, wherein the process of using the flow cell device comprises removing a residual amount of the first reagent or the second reagent from the open landing area through a clean outlet of the flow cell device.

352. The method of claim 351, wherein the clean outlet is configured to allow residual amounts of the first reagent on the open landing area to flow through the clean outlet.

353. The method according to any one of claims 231 to 352, wherein the flow cell system comprises the flow cell device, and wherein the flow cell system further comprises a fluid control device, the fluid control device comprising: (a) a first pump, the first pump being fluidly connected to the clean outlet, wherein the first pump or the second pump is fluidly connected to the outlet; and (b) A dispenser configured to openly dispense the one or more reagents into the inlet.

354. The method of claim 353, wherein the first pump is configured to clean the open landing area by displacing a residual amount of the first reagent away from the open landing area to flow through the clean outlet.

355. The method of claim 343, wherein the process further comprises: At least a portion of the first reagent is removed from at least a portion of the one or more channels by driving fluid through the air gap between the inlet and through at least a portion of the one or more channels.

356. The method according to any one of claims 351 to 355, wherein the residual amount of the first reagent in the open landing area comprises the meniscus of the first reagent.

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