Microfluidic chip, spatial omics sequencing chip and encoding array position calibration method

Abstract

The present application relates to the technical field of chip sequencing, and discloses a microfluidic chip, a spatial omics sequencing chip and a coding array position calibration method.The microfluidic chip comprises: a first preset number of first coding reagent channels extending along a first direction; a second preset number of second coding reagent channels extending along a second direction; the first direction and the second direction are arranged in a cross manner; a first reference frame reagent channel is arranged on one side of the first coding reagent channel and is parallel to the first coding reagent channel; a second reference frame reagent channel is arranged on the other side of the first coding reagent channel and is parallel to the first coding reagent channel; a third reference frame reagent channel is arranged on one side of the second coding reagent channel and is parallel to the second coding reagent channel; and a fourth reference frame reagent channel is arranged on the other side of the second coding reagent channel and is parallel to the second coding reagent channel.The present application realizes accurate calibration of the coding array position.

Description

Technical Field

[0001] This invention relates to the field of chip sequencing technology, specifically to microfluidic chips, spatial omics sequencing chips, and methods for locating coding arrays. Background Technology

[0002] The spatial gene expression chip has a reference frame that marks the location of the Spot (a single spatial region that captures messenger ribonucleic acid) capture array. By associating tissue location with HE scan images, the distribution map of Spot-captured genes can be aligned with the HE scan images to assess the accuracy of the experiment.

[0003] Organomics sequencing chips, also known as coding chips, are fabricated by sequentially injecting coding regions in the X and Y directions vertically onto a functionalized substrate using microfluidic chips. However, existing methods for fabricating coding chips lack a reference frame, making it impossible to accurately calibrate the position of the coding array and effectively align the Spot Capture Gene Distribution Map with the HE scan map. This results in an inability to assess the accuracy of experimental results and a lack of reliability in the obtained results. Summary of the Invention

[0004] In view of this, the present invention provides a spatial omics sequencing chip and a method for calibrating the position of its coding array, in order to solve the problem that coding chips manufactured by existing methods cannot accurately calibrate the position of the coding array.

[0005] In a first aspect, the present invention provides a microfluidic chip, comprising:

[0006] A first preset number of first coded reagent channels extend along a first direction;

[0007] A second preset number of second coded reagent channels extend along a second direction; the first direction and the second direction are intersected.

[0008] The first reference frame reagent channel is located on one side of the first coded reagent channel and is parallel to the first coded reagent channel;

[0009] The second reference frame reagent channel is located on the other side of the first coded reagent channel and is parallel to the first coded reagent channel.

[0010] The third reference frame reagent channel is located on one side of the second coded reagent channel and is parallel to the second coded reagent channel;

[0011] The fourth reference frame reagent channel is located on the other side of the second coded reagent channel and is parallel to the second coded reagent channel; wherein, the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel together form a reference frame, which is used to calibrate the position of the coded array.

[0012] In this embodiment, by setting a first reference frame reagent channel, a second reference frame reagent channel, a third reference frame reagent channel, and a fourth reference frame reagent channel on both sides of the first and second coded reagent channels, the reference frame formed by the first, second, third, and fourth reference frame reagent channels is used as a reference for accurately calibrating the position of the coded array, thereby achieving accurate calibration of the position of the coded array and improving the accuracy and reliability of the experimental results.

[0013] In one alternative implementation, the reference frame is non-centrosymmetric to characterize the orientation of the coding array.

[0014] In this optional embodiment, the orientation of the coding array is determined by a non-centrosymmetric reference frame. When the image is reversed or mirrored, the orientation of the coding array can be identified by the non-centrosymmetric reference frame, thereby accurately restoring the position of the coding array and achieving accurate calibration of the orientation and position of the coding array, thus ensuring the accuracy of the experimental results.

[0015] In one alternative implementation, the first direction intersects the second direction perpendicularly.

[0016] In this optional embodiment, the perpendicular intersection of the first and second directions ensures that deoxyribonucleic acid (DNA) has a well-separated delivery channel in the microfluidic chip.

[0017] In one alternative implementation, the first direction is horizontal and the second direction is vertical.

[0018] In a second aspect, the present invention provides a space omics sequencing chip, characterized in that it comprises a microfluidic chip including the first aspect or any optional embodiment of the first aspect.

[0019] Thirdly, the present invention provides a method for locating the coding array of a space omics sequencing chip, applied to the space omics sequencing chip of the first aspect, the method comprising:

[0020] Obtain the channel parameters of the first coded reagent channel and the second coded reagent channel; wherein the number of the first coded reagent channels is a first preset number, and the number of the second coded reagent channels is a second preset number;

[0021] Based on the channel parameters of the first coded reagent channel and the second coded reagent channel, draw the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel; wherein, the first reference frame reagent channel is located on one side of the first coded reagent channel and is parallel to the first coded reagent channel; the second reference frame reagent channel is located on the other side of the first coded reagent channel and is parallel to the first coded reagent channel; the third reference frame reagent channel is located on one side of the second coded reagent channel and is parallel to the second coded reagent channel; and the fourth reference frame reagent channel is located on the other side of the second coded reagent channel and is parallel to the second coded reagent channel.

[0022] Obtain the reference frame formed by the reagent channels of the first reference frame, the second reference frame, the third reference frame, and the fourth reference frame, and use the reference frame to mark the position of the coding array.

[0023] In this embodiment, by setting a first reference frame reagent channel, a second reference frame reagent channel, a third reference frame reagent channel, and a fourth reference frame reagent channel on both sides of the first and second coding reagent channels, the position of the coding array can be accurately calibrated, making the spatial transcriptome chip product more reliable and ensuring the accuracy of experimental results.

[0024] In one optional implementation, based on the channel parameters of the first coded reagent channel and the second coded reagent channel, a first reference frame reagent channel, a second reference frame reagent channel, a third reference frame reagent channel, and a fourth reference frame reagent channel are drawn, including:

[0025] The channel parameters of the first coded reagent channel and the channel parameters of the second coded reagent channel are compared with a first preset threshold and a second preset threshold, respectively; wherein, the first preset threshold is less than the second preset threshold; the channel parameters of the first coded reagent channel and the channel parameters of the second coded reagent channel include the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel, and the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are the same;

[0026] Based on the comparison results, the channel parameters of the reagent channels in the first, second, third, and fourth reference frames are determined. The channel parameters of the first, second, third, and fourth reference frames include the linewidth and number of the reagent channels in each frame.

[0027] In this optional embodiment, the linewidths and quantities of the first, second, third, and fourth reference frame reagent channels are determined based on the linewidths of the first and second coded reagent channels. This ensures that the flow resistance of the first and second coded reagent channels is consistent with or similar to that of the first, second, third, and fourth reference frame reagent channels. Furthermore, by controlling the linewidths and quantities of the reference frame reagent channels to be slightly different, the reference frames are not centrally symmetrical, and their relative positions to the coding array are accurately guaranteed, thus ensuring the accuracy of the experimental results.

[0028] In one optional implementation, determining the channel parameters of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel based on the comparison results includes:

[0029] When the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are less than or equal to the first preset threshold, the linewidths of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel are set to be the same as the linewidths of the first coded reagent channel and the second coded reagent channel, but the number of the first reference frame reagent channel, the number of the second reference frame reagent channel, the number of the third reference frame reagent channel, and the number of the fourth reference frame reagent channel are different.

[0030] In an optional implementation, determining the channel parameters of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel based on the comparison results further includes:

[0031] When the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are greater than the first preset threshold and less than or equal to the second preset threshold, then any two of the following reference frame reagent channels are set to have the same linewidth: the linewidth of the first reference frame reagent channel, the linewidth of the second reference frame reagent channel, the linewidth of the third reference frame reagent channel, and the linewidth of the fourth reference frame reagent channel. Furthermore, any two of the following reference frame reagent channels have the same number of channels: the number of channels in the first reference frame reagent channel, the number of channels in the second reference frame reagent channel, the number of channels in the third reference frame reagent channel, and the number of channels in the fourth reference frame reagent channel. Specifically, any two reference frame reagent channels with the same linewidth have different numbers of channels, and any two reference frame reagent channels with the same number of channels have different linewidths.

[0032] In an optional implementation, determining the channel parameters of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel based on the comparison results further includes:

[0033] When the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are greater than the second preset threshold, the number of reagent channels in the first reference frame, the second reference frame, the third reference frame, and the fourth reference frame are set to be the same, and the linewidths of the first reference frame reagent channels, the second reference frame reagent channels, the third reference frame reagent channels, and the fourth reference frame reagent channels are different. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram of a microfluidic chip according to an embodiment of the present invention;

[0036] Figure 2 This is a schematic diagram showing a first reference frame reagent channel and a second reference frame reagent channel arranged on both sides of a first coded reagent channel according to an embodiment of the present invention;

[0037] Figure 3 This is a schematic diagram showing that a third reference frame reagent channel and a fourth reference frame reagent channel are provided on both sides of the second coded reagent channel according to an embodiment of the present invention;

[0038] Figure 4 This is a schematic diagram of a control strategy with the same quantity but different line widths according to an embodiment of the present invention;

[0039] Figure 5 This is a schematic diagram of a control strategy with consistent line widths but different numbers according to an embodiment of the present invention;

[0040] Figure 6 This is a schematic diagram of a control strategy that combines quantity and linewidth according to an embodiment of the present invention;

[0041] Figure 7 This is a flowchart illustrating a method for calibrating the coding array position of a spatial omics sequencing chip according to an embodiment of the present invention.

[0042] Figure 8 This is a flowchart illustrating another method for determining the coding array position of a spatial omics sequencing chip according to an embodiment of the present invention;

[0043] Figure 9 This is a schematic diagram of the hardware structure of an electronic device according to an embodiment of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] The 10X Genomics spatial gene expression chip (a comprehensive single-cell sequencing technology platform) has a reference frame for calibrating the location of the spot capture array. By associating tissue location with HE scan images, it is possible to align the spot capture gene distribution map with the HE scan image to assess the accuracy of the experiment. Tissue spatial omics sequencing chips based on NGS barcoding (next-generation sequencing encoding) technology, also called coding chips, can be fabricated by sequentially introducing X and Y direction vertical cross-samples onto a functionalized substrate using microfluidic chips. However, coding chips fabricated by existing methods lack a reference frame similar to the 10X Genomics spatial gene expression chip, making it impossible to accurately calibrate the location of the coding array and effectively align the spot capture gene distribution map with the HE scan image. This results in the inability to assess the accuracy of experimental results and a lack of reliability in the obtained experimental results.

[0046] The 10X Genomics spatial gene expression chip user manual shows a bright-field visible rectangular reference frame. The patterns corresponding to the four sides and four vertices of the rectangular reference frame are all different. The spot array, which is not visible in the bright field, is located inside the rectangular reference frame, and its positional relationship with the reference frame is fixed. In actual use, the HE scan image associates the reference frame with the position of the tissue section, while the Spot capture gene distribution image associates the reference frame with the tissue gene distribution image. By aligning the reference frame on the tissue HE scan image with the Spot capture gene distribution image, it is possible to analyze whether the tissue section and the tissue gene distribution accurately correspond, thereby assessing the accuracy of the experimental results.

[0047] In existing spatial transcriptome sequencing technologies, X- and Y-direction cross-coding is performed on functionalized substrates to create coding arrays. However, the resulting coding arrays are not visible in the bright field and lack a visible reference frame for calibrating the position of the coding arrays. This results in the acquisition of spot-captured gene distribution maps and tissue HE scans lacking an accurate reference for calibrating their positions, ultimately leading to a lack of reliability in the experimental results.

[0048] This embodiment provides a microfluidic chip, such as Figure 1 As shown, it includes:

[0049] The first preset number of first coded reagent channels 1 extend along the first direction.

[0050] The second preset number of second coded reagent channels 2 extend along the second direction; the first direction and the second direction are intersected.

[0051] The first reference frame reagent channel 3 is located on one side of the first coded reagent channel 1 and is parallel to the first coded reagent channel 1.

[0052] The second reference frame reagent channel 4 is located on the other side of the first coded reagent channel 1 and is parallel to the first coded reagent channel 1.

[0053] The third reference frame reagent channel 5 is located on one side of the second coded reagent channel 2 and is parallel to the second coded reagent channel 2.

[0054] The fourth reference frame reagent channel 6 is located on the other side of the second coded reagent channel 2 and is parallel to the second coded reagent channel 2; wherein, the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5 and the fourth reference frame reagent channel 6 together form a reference frame, which is used to calibrate the position of the coded array.

[0055] Specifically, the first direction and the second direction intersect perpendicularly; the first direction is horizontal, and the second direction is vertical.

[0056] Furthermore, such as Figure 2 As shown, a first reference frame reagent channel 3 (A, a) is set on one side (Y1) of the first coded reagent channel 1, where A represents the number of first reference frame reagent channels 3 and a represents the line width of the first reference frame reagent channel 3; a second reference frame reagent channel 4 (B, b) is set on the other side (Yn) of the first coded reagent channel 1, where B represents the number of second reference frame reagent channels 4 and b represents the line width of the second reference frame reagent channel 4; as Figure 3 As shown, a third reference frame reagent channel 5 (C, c) is set on one side (X1) of the second coded reagent channel 2, where C represents the number of third reference frame reagent channels 5 and c represents the line width of the third reference frame reagent channel 5; a fourth reference frame reagent channel 6 (D, d) is set on the other side (Xn) of the second coded reagent channel 2, where D represents the number of fourth reference frame reagent channels 6 and d represents the line width of the fourth reference frame reagent channel 6.

[0057] Specifically, the reference frame is non-centrally symmetric to characterize the orientation of the coding array.

[0058] Furthermore, to ensure that the channel parameters (including quantity and linewidth) of the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5, and the fourth reference frame reagent channel 6 are not completely consistent, the following control strategies are determined: consistent quantity, different linewidth; consistent linewidth, different quantity; mixed quantity and linewidth; thus, the positional relationship between the reference frames drawn based on the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5, and the fourth reference frame reagent channel 6 and the coding array is accurate and unique.

[0059] Furthermore, based on the linewidths of the first coded reagent channel 1 and the second coded reagent channel 2, the channel parameters of the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5, and the fourth reference frame reagent channel 6 are determined: when the linewidth is ≤25um (micrometers), "consistent linewidth, different quantity" is selected (i.e., a=b=c=d, A, B, C, and D are all different); when 25um<linewidth≤50um, "mixed quantity and linewidth" is selected (for example, quantity A=C, B=D; linewidth a=b, c=d, the linewidths a and c corresponding to the same quantity of A and C are different, and the linewidths b and d corresponding to the same quantity of B and D are different); when 50um<linewidth, "consistent quantity, different linewidth" is selected (i.e., A=B=C=D, a, b, c, and d are all different).

[0060] Furthermore, because the parameters controlling the reagent channels of the reference frame are not completely consistent, the four sides (number and line width) of the drawn reference frame are different, and the vertex patterns where the four sides intersect are also different (i.e., the reference frame is not centrally symmetric). This allows for accurate identification of which reagent channel drew each of the four sides of the reference frame. In other words, when the image is reversed or mirrored, the direction of the coding array (i.e., the start and end points of the coding sequence) can be determined through the non-centrally symmetric reference frame. Since the relative positional relationship between the reagent channels of the reference frame and the coding reagent channels on the microfluidic chip is known, the reference frame edges drawn by the reagent channels have the same positional relationship as the coding sequence processed by the coding reagent channels. Therefore, the position of the coding sequence can be reconstructed from the reference frame, thereby accurately calibrating the position of the coding array, as detailed below:

[0061] a. Obtain a reference frame and determine which of the four sides of the reference frame is drawn by the outer reference frame reagent channel of the coded reagent channel;

[0062] b. Determine the positions of the X1-Xn and Y1-Ym sequences within the reference frame based on the positional relationship between the reagent channels in the reference frame and the coded reagent channels on the microfluidic chip.

[0063] c. The X1-Xn and Y1-Ym sequences intersect to form a unique array, which is the encoding array.

[0064] The microfluidic chip provided in this embodiment controls the parameters of the reference frame reagent channels to be not completely consistent, making the reference frame non-centrosymmetric and its relative position to the coding array accurate. This achieves the same effect as the reference frame of the 10XGenomics spatial gene expression chip, making NGS-barcoding-based spatial transcriptome chip products more reliable. Furthermore, the linewidths and numbers of the first, second, third, and fourth reference frame reagent channels are determined based on the linewidths of the first and second coding reagent channels, ensuring that the flow resistance of the first and second coding reagent channels is consistent with or similar to that of the first, second, third, and fourth reference frame reagent channels, thus guaranteeing the reliability of the processing and the accuracy of the experimental results.

[0065] The following specific examples illustrate the process of calibrating the coding sequence position of a microfluidic chip.

[0066] Example 1:

[0067] like Figure 4As shown, assuming the linewidths of the first coded reagent channel 1 and the second coded reagent channel 2 are >50µm, the number of reagent channels 3, 4, 5, and 6 in the first reference frame, the second reference frame, the third reference frame, and the fourth reference frame are kept consistent, i.e., A = B = C = D, and the number is set to 1; the linewidths are controlled to be different, i.e., a, b, c, and d are not equal, and a = 50µm, b = 100µm, c = 25µm, and d = 75µm are set.

[0068] The channel parameters of the first reference frame reagent channel 3 are expressed as (1, 50um), that is, the number of reagent channels in the first reference frame is 1 and the line width is 50um. The channel parameters of the second reference frame reagent channel 4 are expressed as (1, 100um). The channel parameters of the third reference frame reagent channel 5 are expressed as (1, 25um). The channel parameters of the fourth reference frame reagent channel 6 are expressed as (1, 75um).

[0069] Based on the positional relationship between the reagent channels of each reference frame and each coded reagent channel on the microfluidic chip, the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5 and the fourth reference frame reagent channel 6 are determined to form a reference frame together;

[0070] The reagent channel 3 of the first reference frame (1, 50 μm) is used as the calibration reference for the starting point Y1 of the vertical coding sequence in the coding array, and the reagent channel 4 of the second reference frame (1, 100 μm) is used as the calibration reference for the ending point Ym of the vertical coding sequence in the coding array, thereby calibrating the vertical sequence of the coding array as Y1 to Ym.

[0071] The reagent channel 5 of the third reference frame (1, 25um) is used as the calibration reference for the starting point X1 of the transverse coding sequence in the coding array, and the reagent channel 5 of the third reference frame (1, 75um) is used as the calibration reference for the ending point Xn of the transverse sequence in the coding array, thereby calibrating the transverse sequence of the coding array as X1 to Xn.

[0072] The final calibrated coding array is located in the area within the reference frame formed by the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5, and the fourth reference frame reagent channel 6, and its coding sequence is (X1, Y1)-(Xn, Ym).

[0073] Example 2:

[0074] like Figure 5As shown, assuming the linewidths of the first coded reagent channel 1 and the second coded reagent channel 2 are ≤25um, the linewidths of the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5, and the fourth reference frame reagent channel 6 are kept consistent, i.e., a=b=c=d, and the linewidth value is taken as 25um; the control quantities are different, i.e., A, B, C, and D are not equal, and the values ​​are taken as A=2, B=4, C=1, and D=3;

[0075] The channel parameters of reagent channel 3 in the first reference box are (2, 25 μm), the channel parameters of reagent channel 4 in the second reference box are (4, 25 μm), the channel parameters of reagent channel 5 in the third reference box are (1, 25 μm), and the channel parameters of reagent channel 6 in the fourth reference box are (3, 25 μm).

[0076] Based on the positional relationship between the reagent channels of each reference frame and each coded reagent channel on the microfluidic chip, the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5 and the fourth reference frame reagent channel 6 are determined to form a reference frame together;

[0077] The reagent channel 3 of the first reference frame (2, 25um) is used as the calibration reference for the starting point Y1 of the vertical coding sequence in the coding array, and the reagent channel 4 of the second reference frame (4, 25um) is used as the calibration reference for the ending point Ym of the vertical coding sequence in the coding array. Thus, the vertical sequence of the coding array is calibrated as Y1 to Ym.

[0078] The reagent channel 5 of the third reference frame (1, 25um) is used as the calibration reference for the starting point X1 of the transverse coding sequence in the coding array, and the reagent channel 5 of the third reference frame (3, 25um) is used as the calibration reference for the ending point Xn of the transverse sequence in the coding array, thereby calibrating the transverse sequence of the coding array as X1 to Xn.

[0079] The final calibrated coding array is located in the area within the reference frame formed by the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5, and the fourth reference frame reagent channel 6, and its coding sequence is (X1, Y1)-(Xn, Ym).

[0080] Example 3:

[0081] like Figure 6 As shown, assuming the linewidth of the first coded reagent channel 1 and the second coded reagent channel 2 is 25um < linewidth ≤ 50um, the linewidth and quantity of the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5 and the fourth reference frame reagent channel 6 are mixed, that is, the quantity A = C = 1, B = D = 2, and the linewidth a = b = 50um, c = d = 100um;

[0082] The channel parameters of reagent channel 3 in the first reference box are (1, 50 μm), the channel parameters of reagent channel 4 in the second reference box are (2, 50 μm), the channel parameters of reagent channel 5 in the third reference box are (1, 100 μm), and the channel parameters of reagent channel 6 in the fourth reference box are (2, 100 μm).

[0083] Based on the positional relationship between the reagent channels of each reference frame and each coded reagent channel on the microfluidic chip, the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5 and the fourth reference frame reagent channel 6 are determined to form a reference frame together;

[0084] The reagent channel 3 of the first reference frame (1, 50um) is used as the calibration reference for the starting point Y1 of the vertical coding sequence in the coding array, and the reagent channel 4 of the second reference frame (2, 50um) is used as the calibration reference for the ending point Ym of the vertical coding sequence in the coding array, thereby calibrating the vertical sequence of the coding array as Y1 to Ym.

[0085] The reagent channel 5 of the third reference frame (1, 100um) is used as the calibration reference for the starting point X1 of the transverse coding sequence in the coding array, and the reagent channel 5 of the third reference frame (2, 100um) is used as the calibration reference for the ending point Xn of the transverse sequence in the coding array, thereby calibrating the transverse sequence of the coding array as X1 to Xn.

[0086] The final calibrated coding array is located in the area within the reference frame formed by the first reference frame reagent channel 3, the second reference frame reagent channel 4, the third reference frame reagent channel 5, and the fourth reference frame reagent channel 6, and its coding sequence is (X1, Y1)-(Xn, Ym).

[0087] This embodiment also provides a space omics sequencing chip, including a microfluidic chip as claimed in any one of claims 1 to 4.

[0088] Specifically, the microfluidic chip material includes glass, silicon wafer, or polymer material; polymer material includes polydimethylsiloxane (PDMS), polyurethane, epoxy resin, polymethyl methacrylate (PMMA), polycarbonate (PC), cyclic olefin copolymer (COC), polystyrene (PS), polyethylene (PE), fluoroplastics, and glass; the microfluidic chip material can be one or more of the above materials.

[0089] Furthermore, depending on the material and structure, the microfluidic chip fabrication method can select one or more of different methods such as photolithography, CNC machining, casting, injection molding, laser engraving, plasma etching, and wet etching.

[0090] Furthermore, the reagents used to draw the reference frame include one or more of gold nanoparticles, colored silica microspheres, or common colored dyes.

[0091] Furthermore, the substrate material used for encoding includes one of the following: silicon wafer, glass slide, cover glass, indium tin oxide (ITO) conductive glass, and polymethyl methacrylate (PMMA) sheet.

[0092] Furthermore, the modification of functional groups on the substrate surface can be carried out using physical methods such as etching, vapor deposition, and spin coating, as well as chemical methods such as in-situ synthesis, covalent coupling, molecular assembly, and affinity linkage, depending on the requirements for connection with the 5' end groups of oligonucleotides.

[0093] This embodiment also provides a method for locating the coding array of a space omics sequencing chip, applied to the aforementioned space omics sequencing chip, such as... Figure 7 As shown, the process includes the following steps:

[0094] Step S701: Obtain the channel parameters of the first coded reagent channel and the second coded reagent channel; wherein, the number of the first coded reagent channels is a first preset number, the number of the second coded reagent channels is a second preset number, the first coded reagent channels extend along a first direction, the second coded reagent channels extend along a second direction, and the first direction and the second direction are intersected.

[0095] Specifically, the first direction and the second direction intersect perpendicularly; the first direction is horizontal, and the second direction is vertical.

[0096] Step S702: Based on the channel parameters of the first coded reagent channel and the second coded reagent channel, draw the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel; wherein, the first reference frame reagent channel is set on one side of the first coded reagent channel and is parallel to the first coded reagent channel; the second reference frame reagent channel is set on the other side of the first coded reagent channel and is parallel to the first coded reagent channel; the third reference frame reagent channel is set on one side of the second coded reagent channel and is parallel to the second coded reagent channel; and the fourth reference frame reagent channel is set on the other side of the second coded reagent channel and is parallel to the second coded reagent channel.

[0097] Specifically, a first reference frame reagent channel (A, a) is set on one side (X1) of the first coded reagent channels X1-Xn, where A represents the number of first reference frame reagent channels and a represents the line width of the first reference frame reagent channel; a second reference frame reagent channel (B, b) is set on the other side (Xn) of the first coded reagent channels X1-Xn, where B represents the number of second reference frame reagent channels and b represents the line width of the second reference frame reagent channel; as shown Figure 3As shown, a third reference frame reagent channel (C, c) is set on one side (Y1) of the second coded reagent channel Y1-Ym, where C represents the number of third reference frame reagent channels and c represents the line width of the third reference frame reagent channel; a fourth reference frame reagent channel (D, d) is set on the other side (Ym) of the second coded reagent channel Y1-Ym, where D represents the number of fourth reference frame reagent channels and d represents the line width of the fourth reference frame reagent channel.

[0098] Step S703: Obtain the reference frame formed by the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel, and use the reference frame to calibrate the position of the coding array.

[0099] Specifically, the reference frame is non-centrally symmetric to characterize the orientation of the coding array.

[0100] This embodiment provides a method for calibrating the coding array position of a spatial omics sequencing chip. By setting a first reference frame reagent channel, a second reference frame reagent channel, a third reference frame reagent channel, and a fourth reference frame reagent channel on both sides of the first and second coding reagent channels, the position of the coding array can be accurately calibrated, making the NGS-barcoding-based spatial transcriptome chip products more reliable and ensuring the accuracy of experimental results.

[0101] This embodiment provides a method for locating the coding array of a space omics sequencing chip, which is applied to the aforementioned space omics sequencing chip. Figure 8 This is a flowchart of a method for determining the coding array position of a space omics sequencing chip according to an embodiment of the present invention, as shown below. Figure 8 As shown, the process includes the following steps:

[0102] Step S801: Obtain the channel parameters of the first coded reagent channel and the second coded reagent channel; wherein the number of the first coded reagent channels is a first preset number, and the number of the second coded reagent channels is a second preset number. For details, please refer to [link to relevant documentation]. Figure 7 Step S701 of the illustrated embodiment will not be described again here.

[0103] Step S802: Based on the channel parameters of the first coded reagent channel and the second coded reagent channel, draw the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel; wherein, the first reference frame reagent channel is set on one side of the first coded reagent channel and is parallel to the first coded reagent channel; the second reference frame reagent channel is set on the other side of the first coded reagent channel and is parallel to the first coded reagent channel; the third reference frame reagent channel is set on one side of the second coded reagent channel and is parallel to the second coded reagent channel; and the fourth reference frame reagent channel is set on the other side of the second coded reagent channel and is parallel to the second coded reagent channel.

[0104] Specifically, step 802 above includes:

[0105] Step S8021: Compare the channel parameters of the first coded reagent channel and the second coded reagent channel with a first preset threshold and a second preset threshold, respectively; wherein, the first preset threshold is less than the second preset threshold; the channel parameters of the first coded reagent channel and the second coded reagent channel include the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel, and the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are the same.

[0106] Step S8022: Based on the comparison results, determine the channel parameters of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel; wherein, the channel parameters of the first reference frame reagent channel include the linewidth and number of the first reference frame reagent channel, the channel parameters of the second reference frame reagent channel include the linewidth and number of the second reference frame reagent channel, the channel parameters of the third reference frame reagent channel include the linewidth and number of the third reference frame reagent channel, and the channel parameters of the fourth reference frame reagent channel include the linewidth and number of the fourth reference frame reagent channel; the channel parameters of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel are used to determine the orientation of the coding array.

[0107] Specifically, the channel parameters (including quantity and linewidth) of the reagent channels of the first, second, third, and fourth reference boxes are not completely consistent. The following control strategies are determined: consistent quantity, different linewidth; consistent linewidth, different quantity; mixed quantity and linewidth; thus, the positional relationship between the reference boxes drawn based on the reagent channels of the first, second, third, and fourth reference boxes and the coding array is accurate and unique.

[0108] Furthermore, based on the linewidths of the first and second coded reagent channels, the channel parameters of the first, second, third, and fourth reference frame reagent channels are determined: when the linewidth is ≤25µm, "consistent linewidth, different quantity" is selected (i.e., a=b=c=d, A, B, C, and D are all different); when 25µm<linewidth≤50µm, "mixed quantity and linewidth" is selected (for example, quantity A=C, B=D; linewidth a=b, c=d, the linewidths a and c corresponding to the same quantity of A and C are different, and the linewidths b and d corresponding to the same quantity of B and D are different); when 50µm<linewidth, "consistent quantity, different linewidth" is selected (i.e., A=B=C=D, a, b, c, and d are all different).

[0109] Furthermore, because the parameters controlling the reagent channels of the reference frame are not completely consistent, the four sides (number and line width) of the drawn reference frame are all different, and the vertex patterns where the four sides intersect are also different (i.e., the reference frame is not centrosymmetric). Therefore, it is possible to accurately identify which of the four reference frame reagent channels drew each of the reference frame's four sides. That is, when the image is reversed or mirrored, the direction of the coding array (i.e., the start and end points of the coding sequence) can be determined through the non-centrosymmetric reference frame. Since the relative positional relationship between the reference frame reagent channels and the coded reagent channels on the microfluidic chip is known, the reference frame... The reference frame edges drawn by the reagent channel have the same positional relationship as the encoded sequence processed by the coded reagent channel. Therefore, the position of the encoded sequence is reconstructed from the reference frame, thus accurately calibrating the position of the encoded array. Specifically, a reference frame is obtained, and it is determined which of the four edges of the reference frame were drawn by which coded reagent channel. Based on the positional relationship between the reference frame reagent channel and the coded reagent channel on the microfluidic chip, the positions of the X1-Xn and Y1-Ym sequences within the reference frame are determined. The X1-Xn and Y1-Ym sequences intersect to form a unique array, which is the encoded array.

[0110] In some optional implementations, step S8022 above includes:

[0111] Step a1: When the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are less than or equal to the first preset threshold, the linewidths of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel are set to be the same as the linewidths of the first coded reagent channel and the second coded reagent channel, but the number of the first reference frame reagent channel, the number of the second reference frame reagent channel, the number of the third reference frame reagent channel, and the number of the fourth reference frame reagent channel are different.

[0112] Step a2: When the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are greater than the first preset threshold and less than or equal to the second preset threshold, then any two of the following reference frame reagent channels are set to have the same linewidth: the linewidth of the first reference frame reagent channel, the linewidth of the second reference frame reagent channel, the linewidth of the third reference frame reagent channel, and the linewidth of the fourth reference frame reagent channel. Any two of the following reference frame reagent channels have the same number of channels: the number of channels in the first reference frame, the number of channels in the second reference frame, the number of channels in the third reference frame, and the number of channels in the fourth reference frame. Wherein, any two reference frame reagent channels with the same linewidth have different numbers of channels, and any two reference frame reagent channels with the same number of channels have different linewidths.

[0113] Step a3: When the linewidth of the first coded reagent channel and the linewidth of the second coded reagent channel are greater than the second preset threshold, the number of reagent channels in the first reference frame, the second reference frame, the third reference frame, and the fourth reference frame are set to be the same, and the linewidths of the first reference frame reagent channels, the second reference frame reagent channels, the third reference frame reagent channels, and the fourth reference frame reagent channels are different.

[0114] Step S803: Obtain the reference frame region formed by the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel, and the fourth reference frame reagent channel, and use the reference frame region to calibrate the position of the coding array. For details, please refer to [link to relevant documentation]. Figure 7 Step S803 of the illustrated embodiment will not be described again here.

[0115] The microfluidic chip provided in this embodiment, by setting a first reference frame reagent channel, a second reference frame reagent channel, a third reference frame reagent channel, and a fourth reference frame reagent channel on both sides of the first and second coding reagent channels, can accurately calibrate the position of the coding array. Furthermore, by controlling the parameters of the reference frame reagent channels to be not completely consistent, the reference frames are non-centrosymmetric, and their relative positions with the coding array are accurate, achieving the same effect as the reference frames of 10X Genomics spatial gene expression chips. This makes NGS-barcoding-based spatial transcriptome chip products more reliable. Finally, the linewidths and numbers of the first, second, third, and fourth reference frame reagent channels are determined based on the linewidths of the first and second coding reagent channels, ensuring that the flow resistance of the first and second coding reagent channels is consistent with or similar to that of the first, second, third, and fourth reference frame reagent channels, thus guaranteeing the reliability of the processing and the accuracy of the experimental results.

[0116] This invention also provides an electronic device, please refer to [link / reference]. Figure 9 , Figure 9 This is a schematic diagram of the structure of an electronic device provided in an optional embodiment of the present invention, such as... Figure 9 As shown, the electronic device includes one or more processors 10, memory 20, and interfaces for connecting the components, including high-speed interfaces and low-speed interfaces. The components communicate with each other via different buses and can be mounted on a common motherboard or otherwise as required. The processors can process instructions executed within the electronic device, including instructions stored in or on memory to display graphical information of a GUI on external input / output devices (such as display devices coupled to the interfaces). In some alternative implementations, multiple processors and / or multiple buses can be used with multiple memories and multiple memory modules, if desired. Similarly, multiple electronic devices can be connected, each providing some of the necessary operations (e.g., as a server array, a group of blade servers, or a multiprocessor system). Figure 9 Take a processor 10 as an example.

[0117] Processor 10 may be a central processing unit, a network processor, or a combination thereof. Processor 10 may further include a hardware chip. The hardware chip may be an application-specific integrated circuit (ASIC), a programmable logic device (PLD), or a combination thereof. The programmable logic device may be a complex programmable logic device (CAMP), a field-programmable gate array (FPGA), a general-purpose array logic (GPA), or any combination thereof.

[0118] The memory 20 stores instructions executable by at least one processor 10 to cause at least one processor 10 to perform the method shown in the above embodiments.

[0119] The memory 20 may include a program storage area and a data storage area. The program storage area may store the operating system and applications required for at least one function; the data storage area may store data created based on the use of the electronic device. Furthermore, the memory 20 may include high-speed random access memory and may also include non-transitory memory, such as at least one disk storage device, flash memory device, or other non-transitory solid-state storage device. In some alternative embodiments, the memory 20 may optionally include memory remotely located relative to the processor 10, and these remote memories may be connected to the electronic device via a network. Examples of such networks include, but are not limited to, the Internet, intranets, local area networks, mobile communication networks, and combinations thereof.

[0120] The memory 20 may include volatile memory, such as random access memory; the memory may also include non-volatile memory, such as flash memory, hard disk or solid-state drive; the memory 20 may also include a combination of the above types of memory.

[0121] The electronic device also includes a communication interface 30 for communicating with other devices or communication networks.

[0122] This invention also provides a computer-readable storage medium. The methods described above according to embodiments of the invention can be implemented in hardware or firmware, or implemented as computer code that can be recorded on a storage medium, or implemented as computer code downloaded via a network and originally stored on a remote storage medium or a non-transitory machine-readable storage medium and then stored on a local storage medium. Thus, the methods described herein can be processed by software stored on a storage medium using a general-purpose computer, a dedicated processor, or programmable or dedicated hardware. The storage medium can be a magnetic disk, optical disk, read-only memory, random access memory, flash memory, hard disk, or solid-state drive, etc.; further, the storage medium can also include combinations of the above types of memory. It is understood that computers, processors, microprocessor controllers, or programmable hardware include storage components capable of storing or receiving software or computer code, which, when accessed and executed by the computer, processor, or hardware, implements the methods shown in the above embodiments.

[0123] Although embodiments of the invention have been described in conjunction with the accompanying drawings, those skilled in the art can make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations all fall within the scope defined by the appended claims.

Claims

1. A method for encoding array position calibration of a spatial omics sequencing chip, comprising: The application is applied to a spatial omics sequencing chip, and the spatial omics sequencing chip comprises a microfluidic chip, and the microfluidic chip comprises: ​ A first preset number of first coded reagent channels extending in a first direction; A second preset number of second coded reagent channels extending in a second direction; the first direction and the second direction are arranged in a cross manner; A first reference frame reagent channel is arranged on one side of the first coded reagent channel and is parallel to the first coded reagent channel; A second reference frame reagent channel is arranged on the other side of the first coded reagent channel and is parallel to the first coded reagent channel; A third reference frame reagent channel is arranged on one side of the second coded reagent channel and is parallel to the second coded reagent channel; A fourth reference frame reagent channel is arranged on the other side of the second coded reagent channel and is parallel to the second coded reagent channel; wherein the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel form a reference frame, and the reference frame is used for calibrating the position of a coded array; The method comprises: Obtaining channel parameters of the first coded reagent channels and the second coded reagent channels; wherein the number of the first coded reagent channels is a first preset number, and the number of the second coded reagent channels is a second preset number; According to the channel parameters of the first coded reagent channels and the second coded reagent channels, drawing the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel; wherein the first reference frame reagent channel is arranged on one side of the first coded reagent channel and is parallel to the first coded reagent channel; the second reference frame reagent channel is arranged on the other side of the first coded reagent channel and is parallel to the first coded reagent channel; the third reference frame reagent channel is arranged on one side of the second coded reagent channel and is parallel to the second coded reagent channel; and the fourth reference frame reagent channel is arranged on the other side of the second coded reagent channel and is parallel to the second coded reagent channel; Obtaining the reference frame formed by the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel, and calibrating the position of the coded array by using the reference frame; According to the channel parameters of the first coded reagent channels and the second coded reagent channels, drawing the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel, comprises: Comparing the channel parameters of the first coded reagent channels and the channel parameters of the second coded reagent channels with a first preset threshold and a second preset threshold respectively; wherein the first preset threshold is smaller than the second preset threshold; the channel parameters of the first coded reagent channels and the channel parameters of the second coded reagent channels comprise the line width of the first coded reagent channels and the line width of the second coded reagent channels, and the line width of the first coded reagent channels and the line width of the second coded reagent channels are the same; determine the channel parameters of the first, second, third and fourth reference frame reagent channels based on the comparison result, wherein the channel parameters of the first reference frame reagent channel include the line width and number of the first reference frame reagent channel, the channel parameters of the second reference frame reagent channel include the line width and number of the second reference frame reagent channel, the channel parameters of the third reference frame reagent channel include the line width and number of the third reference frame reagent channel, and the channel parameters of the fourth reference frame reagent channel include the line width and number of the fourth reference frame reagent channel; and the channel parameters of the first, second, third and fourth reference frame reagent channels are used to determine the direction of the coding array.

2. The method of claim 1, wherein, The reference frame is non-centrosymmetric to represent the direction of the coding array.

3. The method of claim 1, wherein, The first direction and the second direction are perpendicular to each other.

4. The method according to claim 1 or 3, characterized in that, The first direction is a transverse direction, and the second direction is a longitudinal direction.

5. The method of claim 1, wherein, The determination of the channel parameters of the first, second, third and fourth reference frame reagent channels based on the comparison result includes: When the line width of the first coding reagent channel and the line width of the second coding reagent channel are less than or equal to the first preset threshold, the line width of the first reference frame reagent channel, the line width of the second reference frame reagent channel, the line width of the third reference frame reagent channel and the line width of the fourth reference frame reagent channel are set to be the same as the line width of the first coding reagent channel and the line width of the second coding reagent channel, and the number of the first reference frame reagent channel, the number of the second reference frame reagent channel, the number of the third reference frame reagent channel and the number of the fourth reference frame reagent channel are different.

6. The method of claim 5, wherein, The determination of the channel parameters of the first, second, third and fourth reference frame reagent channels based on the comparison result further includes: When the line width of the first coding reagent channel and the line width of the second coding reagent channel are greater than the first preset threshold and less than or equal to the second preset threshold, the line width of any two of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel is set to be the same, and the number of any two of the first reference frame reagent channel, the second reference frame reagent channel, the third reference frame reagent channel and the fourth reference frame reagent channel is set to be the same; wherein the number of the two reference frame reagent channels with the same line width is different, and the line width of the two reference frame reagent channels with the same number is different.

7. The method of claim 5, wherein, The determining the channel parameters of the first reference frame reagent channel, the channel parameters of the second reference frame reagent channel, the channel parameters of the third reference frame reagent channel and the channel parameters of the fourth reference frame reagent channel based on the comparison result further comprises: When the line width of the first encoding reagent channel and the line width of the second encoding reagent channel are greater than the second preset threshold, the number of the first reference frame reagent channel, the number of the second reference frame reagent channel, the number of the third reference frame reagent channel and the number of the fourth reference frame reagent channel are set to be the same, and the line width of the first reference frame reagent channel, the line width of the second reference frame reagent channel, the line width of the third reference frame reagent channel and the line width of the fourth reference frame reagent channel are different.

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