High-resolution space omics detection method for tissue sample
阅读说明：本技术 一种用于组织样本的高分辨率空间组学检测方法 (High-resolution space omics detection method for tissue sample ) 是由 张俊虎 梁重阳 于年祚 金正洋 杨柏 于 2021-08-06 设计创作，主要内容包括：本发明适用于一种组织样本的高分辨率空间组学检测装置、系统和方法,提供了一种用于组织样本的高分辨率空间组学检测的装置、系统和方法,分别包含：一种带有可容纳微载体的微井反应室阵列的玻片,一种修饰核酸分子标识符的方法和一种在捕获组织样本空间组学信息过程中降低组学信息交叉污染的方法。采用本公开的空间组学检测方法,显著提高了空间组学检测的分辨率,降低了检测成本,同时从根本上降低了空间组学信息的交叉污染。(The invention is suitable for a device, a system and a method for high-resolution space omics detection of tissue samples, and provides a device, a system and a method for high-resolution space omics detection of tissue samples, which respectively comprise: a slide with an array of microwell reaction chambers that can hold microcarriers, a method of modifying nucleic acid molecule identifiers and a method of reducing omic cross-contamination during the capture of tissue sample spatial omic information. By adopting the space omics detection method, the resolution of space omics detection is obviously improved, the detection cost is reduced, and meanwhile, the cross contamination of space omics information is fundamentally reduced.)
1. A high resolution spatial omics detection method for tissue samples, comprising the steps of a: the device comprises a slide with a micro-well array, microcarriers are dispersed in the micro-well array, each micro-well and the microcarriers therein form a micro-reaction chamber, and the device is characterized by further comprising:
step b: transferring a first molecular identifier to a microwell reaction chamber, wherein the first molecular identifier is attached to a microcarrier or a microwell surface, transferring the first molecular identifier to the microwell reaction chamber by using a microchip transfer technology, specifically, aligning parallel micro-channels with a microwell reaction chamber array, and respectively introducing different first molecular identifiers into the channels, wherein the first molecular identifier is attached to the microwell, and the first molecular identifier can be a nucleic acid sequence, namely a first nucleic acid molecular identifier, and the sequence can comprise, from 5 'to 3':
(i) a general domain;
(ii) the first positioning domains are different from one another and can be distinguished from one another, and the sequence information corresponds to the position of the liquid introduced into the pore channel;
(iii) a linking domain, the sequence being for linking between the first and second nucleic acid molecule identifiers;
step c: transferring the second molecular identifier to the microwell reaction chamber by using microchip transfer technology, specifically, realigning the micro channels arranged in parallel to the microwell reaction chamber array in a direction different from the direction of the channels, and respectively introducing different second molecular identifiers into the channels, wherein the first molecular identifier is combined with the second molecular identifier, and the second molecular identifier can be a nucleic acid sequence, namely a second nucleic acid molecular identifier, and the direction from 3 'to 5' can comprise:
a) the connecting domain complementary region can be hybridized with the connecting domain according to the Watson-Crick base complementary pairing principle;
b) the second positioning domains are different from one another and can be distinguished from one another, and the sequence information corresponds to the position of the liquid introduced into the pore channel;
c) molecular markers for providing information on the kind of nucleic acid hybridized to a nucleic acid molecule identifier and distinguishing between types of nucleic acids hybridized to different nucleic acid molecule identifiers, and a second nucleic acid molecule identifier conjugated to the same microcarrier may comprise different molecular markers;
d) a capture domain precursor comprising a nucleic acid sequence for forming a capture domain;
wherein the different first/second nucleic acid molecule identifiers are "different" in that they are different from each other with respect to the molecular identifier attached to the microcarrier of the present disclosure and other molecular identifiers of the present disclosure, and the different first/second molecular identifiers provide spatial location information for omics information captured in the tissue or cell;
step d: incubating the first molecular identifier and the second molecular identifier after hybridization complementation with a reaction mixture, and linking the unique molecular identifier to the microcarrier by means of extension, amplification or ligation, wherein the linking of the unique molecular identifier to the microcarrier comprises linking to the microcarrier according to the present disclosure by any suitable method, and the unique molecular identifier can be a nucleic acid sequence, i.e. a unique nucleic acid molecular identifier, and can include in the 5 'to 3' direction:
1) a general domain;
2) a first positioning field;
3) a connection domain;
4) a second localization domain;
5) molecular marking;
6) a capture domain that can comprise a nucleic acid sequence capable of capturing a nucleic acid sequence, a random sequence, a degenerate capture domain, a sequencing and promoter linker sequence, or a combination thereof, and can also comprise a sequence functionally or structurally similar to a poly-T oligonucleotide sequence;
wherein the unique nucleic acid molecule identifier comprises a nucleic acid sequence of the first nucleic acid molecule identifier that is complementary to the second nucleic acid molecule identifier or a nucleic acid sequence of the complementary nucleic acid sequence that is extended, amplified or linked, and the unique nucleic acid molecule identifier is different from other nucleic acid molecule identifiers linked to microcarriers, nucleic acid sequences associated with cells or tissues, and nucleic acid sequences associated with the present disclosure;
step e: specifically, introducing a solid phase or liquid phase compound into a micro reaction chamber array for storing a microcarrier, attaching a tissue slice to the surface of a microwell array, embedding the tissue sample in the microwell or spreading the tissue sample on the surface of the microwell, wherein the position information of the microcarrier with a specific unique nucleic acid molecule identifier corresponds to the position of a tissue one by one, imaging the tissue sample, covering a porous membrane on the surface of the microwell for preventing the cross contamination among the tissue sample space omics information, adding a tissue permeabilization liquid on the surface of the porous membrane, capturing the nucleic acid sequence of the tissue in the microwell in a limited area by the microcarrier through the unique nucleic acid molecule identifier, and cleaning the surface; incubating the reaction mixed solution in a micro-well array, extending and synthesizing hybrid chains with captured omics information to enable the captured nucleic acid sequences and unique nucleic acid molecule identifiers to form complementary double-stranded nucleic acid sequences, and then amplifying and building a library of the double-stranded nucleic acid sequences; and then, according to the sequence positions of the first positioning domain and the second positioning domain and the imaging detection information, the omics information which is analyzed and is derived from the tissue or the cell sample corresponds to the image space position of the tissue sample according to the position information, thereby obtaining the space omics information of the tissue sample.
2. The method for high resolution spatial omics detection of tissue samples according to claim 1, wherein the microcarriers dispersed in the microwell array comprise microcarriers to which a molecular identifier has been attached, and the method of the present disclosure comprises transferring the microcarriers to which a molecular identifier has been attached to the microwell reaction chamber by any means, and the method comprises attaching the microcarriers in the microwells of the present disclosure to mutually different molecular identifiers by any means, and also comprises attaching the molecular identifier to the interior or surface of the microwell reaction chamber.
3. The high resolution spatial omics detection method for tissue samples according to claim 1, wherein said molecular identifier species comprise nucleic acid sequences, protein molecules, and polysaccharide molecules, and the analysis and detection of nucleic acid molecular identifiers according to the present disclosure is equally applicable to protein and polysaccharide molecular identifiers, i.e., comprises the capture, analysis, and detection of protein and polysaccharide molecules using the methods of the present disclosure.
4. The method for high resolution spatial omics detection of tissue samples according to claim 1, characterized in that said microcarriers are microbeads, gels, polymers, solid phase also comprising any molecular identifier that can be attached, liquid phase carriers and any material that can generate microcarriers known to the person skilled in the art, comprising the interior of the microcarrier, the surface and any other site to which a molecular identifier can be attached.
5. The method for high resolution spatial omics detection of tissue samples according to claim 1, wherein the material of the slide in said kit comprises any material that can be used to prepare topographic structures, the slide comprises at least 1 microwell reaction chamber array, said microwell reaction chamber array comprises at least 1 microwell, and each microwell of said microwell reaction chamber array comprises at least 1 microcarrier.
6. The method for high resolution spatial omics detection of tissue samples according to claim 1, wherein said microwell shape comprises a regular, irregular three-dimensional topographical structure and said microwell reaction chamber has a volume within the range of 0.1fm3-1 cm3。
7. The method for high resolution spatial omics detection of tissue samples according to claim 1, wherein the means for transferring the molecular identifier to the microwell reaction chamber array comprises direct and indirect addition means, and the means for attaching the molecular identifier to the microcarrier comprises but is not limited to physical, chemical, biological modification.
8. The method for high resolution spatial omics detection of tissue samples according to claim 1, wherein the number of the pore channels in the microchannels arranged in parallel is 1 or more.
9. The method for high resolution spaceomics detection of tissue samples as in claim 1, wherein the width of the channels and the width of the space between the channels in the microchannels arranged in parallel are both in the range of 0.1nm to 1000 μm.
10. The high resolution spatial omics detection method for tissue samples according to claim 1, wherein said generic domain comprises:
i. a functional group modification site, a substance capable of binding to the microcarrier or a precursor capable of being activated to form a reactive functional group;
a PCR universal amplification start, which can be complementarily conjugated with a universal primer, for extension or amplification of a nucleic acid molecule; and
a splicing domain for releasing the generated nucleic acid molecule identifier from the microcarrier.
11. The method of claim 1, wherein the method comprises incubating the first nucleic acid molecule identifier and the second nucleic acid molecule identifier after hybridization complementation with a reaction mixture to generate the unique nucleic acid molecule identifier from a microcarrier, wherein the unique nucleic acid molecule identifier comprises in the 5 'to 3' direction: a universal domain, a first localization domain, a linking domain, a second localization domain, a molecular tag, a capture domain, also including: a universal domain, a first localization domain, a second localization domain, a molecular marker, a capture domain, and the reaction mixture may comprise any component that allows for extension, amplification, or ligation of a nucleic acid sequence.
12. The method for high resolution spatial omics detection of tissue samples according to any of claims 1 to 11, wherein the functional regions of said first nucleic acid molecule identifier, said second nucleic acid molecule identifier, and said unique nucleic acid molecule identifier are arranged in a manner that includes, but is not limited to, the order, position, or content listed herein, and wherein one or more of said functional sequences of said molecule identifiers can be arranged in any suitable order or content.
13. The method for high resolution spatial omics detection of tissue samples according to claim 1, wherein said universal domain, universal PCR amplification start, first localization domain, second localization domain, ligation domain complement region, molecular tag, capture domain precursor, and capture domain sequence is at least 1 nucleotide in length.
14. The method for high resolution spatial omics detection of tissue samples according to claim 1, wherein said embedding of the tissue sample into the microwells comprises introducing the tissue sample into the microwells by any external force or by the intrinsic properties of the microwell reaction chamber or by the properties of a solid or liquid phase compound, wherein said solid or liquid phase compound comprises any compound capable of introducing a tissue section into the microwells, and wherein said porous membrane comprises a porous membrane of any material having a pore size ranging from 0.1nm to 100 mm.
15. The method of claim 1, wherein the disclosed method comprises the steps of recovering, pooling, and analyzing the unique nucleic acid molecule identifier or any nucleic acid sequence derived from the unique nucleic acid molecule identifier and the captured nucleic acid as a hybrid, complementary double-stranded nucleic acid sequence, and any nucleic acid sequence converted by the disclosed method from the microcarrier, wherein the steps can be performed on the microcarrier or after recovering the unique nucleic acid molecule identifier with the captured nucleic acid information or the complementary double-stranded nucleic acid sequence from the microcarrier.
16. The high resolution spaceomics detection method for tissue samples of claim 1 wherein the amplification and library methods comprise any known nucleic acid amplification and library methods for amplifying and library target sequence nucleic acid comprising said captured sequence information.
17. The high resolution spatial omics detection method for tissue samples according to claim 1, characterized in that the present disclosure extends to capture and analyze nucleic acid, protein, polysaccharide molecules in tissue, cell, viral samples.
18. The high resolution spatial omics detection method for tissue samples according to claim 1, wherein the tissue sample in the methods of the present disclosure can be a tissue sample or a spatial structure of an organism of any organism.
19. The high resolution spaceomics detection method for tissue samples of claim 1 wherein the tissue samples of the methods of the present disclosure can be any type or kind of tissue sample and the tissue samples of the present disclosure also include any treated or untreated tissue samples.
20. The high resolution spatial omics detection method for tissue samples according to claim 1, characterized in that the method of the present disclosure comprises the use of the method of the present disclosure for obtaining or retrieving omics information unique or independent to any type of single cell or multiple cells.
21. The high resolution spatial omics detection method for tissue samples according to claim 1, characterized in that said method of the present disclosure can be used for spatial transcriptomics studies of tissue sections:
specifically, a solid phase or liquid phase compound is introduced into the micro-reaction chamber array for storing the microcarrier, a tissue slice is attached to the surface of the micro-well array, a tissue sample is embedded into the micro-well, the surface of the micro-well is covered with a porous membrane for preventing cross contamination among tissue sample space omics information, a tissue permeabilizing liquid is added to the surface of the porous membrane, and at the moment, the microcarrier captures mRNA of a limited domain tissue in the micro-well through a capture domain of a unique nucleic acid molecule identifier, and the surface is cleaned; incubating the reverse transcription reaction mixed solution in a micro-well array, extending and synthesizing hybrid chains with captured omics information to enable the captured mRNA and a unique nucleic acid molecule identifier to form cDNA, and then amplifying and establishing a library for the cDNA; and recovering the nucleic acid sequence, analyzing the recovered nucleic acid sequence, and then, according to the first positioning domain information and the second positioning domain information, corresponding the analyzed transcriptomic information from the tissue or cell sample to the spatial position of the tissue sample according to the position information, thereby obtaining the spatial transcription information of the tissue sample.
22. The high resolution spatial omics detection method for tissue samples according to any of claims 1-11, 13-21, wherein the methods of the present disclosure comprise the use of the methods of the present disclosure for any kind of biological omics testing and analysis.
Technical Field
The invention mainly relates to a high-resolution space omics detection device, a system and a method for a tissue sample, in particular to a high-resolution space omics detection method for the tissue sample.
Background
Human tissue is a highly complex system of trillions of cells that vary in kind, time and space, e.g., tissues in different regions of the mammalian brain have different functions and cell types, where detection of spatial heterogeneity of tissues is particularly important. Spatial omics refers to omics studies that are completed on tissue sections and retain spatial information of samples. The space omics can display the gene expression conditions of different areas in the tissue section, reveal the activated signal path in the fine pathological area and complete the mechanism analysis of molecular feature driven pathological features. Space omics complete the technical innovation of pathology digitization combined with pathology imaging, and play an important role in the development of diagnostic markers, drug-resistant sites and targeted drugs, immunotherapy and other emerging fields.
The space omics detection method can be mainly divided into four types, namely a space reconstruction method combining a calculation strategy and an omics experiment, a direct measurement method based on laser microdissection, an in-situ omics method based on a fluorescent probe and image processing and an in-situ capture technology based on oligonucleotide space barcodes.
The spatial reconstruction method obtains the intrinsic gene expression trend of cells and the connection among the cells by integrating single-cell transcriptome data in tissues, but can only present the spatial trend or the overall layout of a specific tissue. The LCM-based spatial omics technology can realize the omics sequencing of single cell resolution, but the technology has low detection flux and is suitable for the detection of the local tissue spatial omics. In-situ omics method based on fluorescent probe and image processing includes two modes of in-situ sequencing (ISS) and in-situ hybridization (ISH), which have outstanding performance in terms of detection resolution, and can realize the spatial omics test at the level of sub-cellular resolution, however, such method has high requirements on detection technology, needs to use a high-sensitivity single-molecule fluorescence imaging system, and the detection needs to go through a complex single-molecule hybridization and image analysis process, which significantly increases the cost and time of omics spatial test, and currently still stays in the application range of laboratories.
The in-situ capture technology based on oligonucleotide space bar codes mainly comprises a microsphere assembly technology adopting fluorescence decoding and an ink-jet spotting method of 10 Xgenomics. In the former method, microspheres decorated with random coding sequences are closely stacked on the surface of a glass slide, and the decoding and positioning of the microsphere sequences are realized by adopting a fluorescence decoding technology, so that the method improves the spatial coding resolution of the captured sequences, but still depends on a complex and expensive single-molecule fluorescence imaging system, and the oligonucleotide capture efficiency of the method is low, and the high-density sequence spatial omics detection is difficult to realize. At present, the most widely used space omics testing method is the Visium product of 10 Xgenomics, the product adopts an ink jet spotting method to prepare a high-flux oligonucleotide capture sequence on the surface of a glass slide, when a tissue sample is attached to the surface of the glass slide, the tissue is subjected to permeabilization treatment, the sequence to be tested of the tissue sample is captured by the oligonucleotide sequence in situ in the process, and space sequencing of the tissue sample is realized after reverse transcription and a second generation sequencing technology (NGS). Compared with the previous methods, the method has the advantages of low sequencing cost, high aging, easy operation and the like, and at present, the 10 xVisium product based on the method is successfully commercialized, and the application range of the method relates to a plurality of important research fields.
However, there are still several important issues to be solved in 10 × Visium: firstly, an oligonucleotide sequence array on the surface of a glass slide is prepared by adopting an ink-jet spotting technology, the highest resolution of the size of the array prepared on the glass slide by the prior art is 55 mu m, each sequence dot matrix corresponds to the space omics information of dozens of cells, which limits the study of single-cell space omics analysis and the interaction mechanism among the cells in a tissue sample and can also cause the omission of important information; secondly, when the oligonucleotide sequence array is prepared by the ink-jet spotting method, the phenomenon of non-uniform spotting can occur, which causes the modification effect between oligonucleotide sequence regions to generate difference, and even causes the condition of spot leakage, namely, samples are not spotted in a target region. Thirdly, when the product is adopted to carry out a sequence capture process, the permeabilizing liquid promotes the sequences in the tissue sample to be exuded and also transversely diffuses the sequences, so that the tissue space sequence information is subjected to cross contamination, and the phenomenon is particularly serious in a high-resolution space omics sequencing process. Fourth, this approach requires full-length synthesis of the oligonucleotide capture sequences separately at each position in the array, and large-scale oligonucleotide synthesis adds cost and design complexity to the product.
Therefore, the defects and limitations of the traditional technology need to be optimized and solved, which is common appeal and general consensus of the industry. Therefore, it is important to develop a tissue sample space omics detection method with high resolution, low cost, less cross contamination and easy operation.
Disclosure of Invention
The embodiment of the invention aims to provide a high-resolution space omics detection method for a tissue sample, and aims to develop a tissue sample space omics detection method which is high in resolution, low in cost, less in cross contamination and easy to operate.
In a first aspect, the present disclosure provides a slide with an array of microwell reaction chambers that can hold microcarriers.
Specifically, a slide containing a micro-well array is constructed, microcarriers are dispersed in the micro-well array, each micro-well and the microcarriers therein form a micro-reaction chamber, a molecular identifier is transferred into the micro-well reaction chamber, the molecular identifier is connected to the surface of the microcarriers or the micro-wells, and the kit is used for tissue space omics research.
Optionally, the material of the slide in the kit comprises any material that can be used to prepare a topographical structure, such as glass, silicon dioxide, polymers.
Alternatively, the method of preparing the microwell reaction chamber includes any method by which a topographical structure may be constructed. For example, a concave feature is formed by etching the inside of a slide by a bottom-up method, growing a concave feature on the upper surface of a slide by a bottom-up method, or forming a concave feature by covering the surface of a slide with a substrate having a micro-well array, a porous membrane.
Optionally, the microwell shape comprises regular and irregular three-dimensional topographical structures, e.g., cylindrical, frustoconical, prismatic topographical structures.
Optionally, the volume in the reaction chamber of the microwell ranges from 0.1fm3-1 cm3Preferably, the microwell reaction chamber has a volume of 10 μm3。
Optionally, the arrangement of the microwell reaction chamber array comprises a regular and irregular arrangement, and preferably, the arrangement of the microwell reaction chamber array is a square array.
Optionally, each microwell of the array of microwell reaction chambers contains at least 1 microcarrier therein. For example, a microwell may contain at least 2, at least 5, at least 10, or at least 100 microcarriers.
Optionally, the microwell reaction chamber array comprises at least 1 microwell. For example, the microwell array may comprise at least 10, at least 100, at least 1000, or at least 10000 microwells.
Optionally, the slide comprises at least 1 array of microwell reaction chambers. For example, a slide can contain at least 2, at least 10, at least 1000, or at least 1000 microwell reaction chamber arrays.
Alternatively, the microcarrier is a microbead, gel or polymer to which the molecular identifier can be attached, and also comprises any solid or liquid phase carrier to which the molecular identifier can be attached and any material from which microcarriers can be generated, as known to those skilled in the art.
Alternatively, the attachment site for attaching the molecular identifier to the microcarrier comprises the interior, surface and any other site to which the molecular identifier can be attached of the microcarrier.
Optionally, the method of transferring the molecular identifier to the array of microwell reaction chambers comprises a direct or indirect addition method, such as inkjet printing, contact printing, to transfer the molecular identifier to the microwell reaction chambers.
Optionally, the types of the molecular identifiers include nucleic acid sequences, protein molecules and other biomolecules, and the analysis and detection of the nucleic acid molecular identifiers in the present disclosure are also applicable to protein and polysaccharide molecular identifiers, that is, the capture, analysis and detection of protein and polysaccharide molecules by the method in the present disclosure are included.
Alternatively, the molecular identifiers transferred into the reaction chambers of the microwells may be molecular identifiers different from each other.
Alternatively, the disclosed method comprises dispersing the microcarriers to which the molecular identifier has been attached in the microwell array, the disclosed method comprises transferring the microcarriers to which the molecular identifier has been attached to the microwell reaction chamber by any means, and the disclosed method comprises attaching the microcarriers in the microwells of the present disclosure to different molecular identifiers by any means, and also comprises attaching the molecular identifiers to the interior or surface of the microwell reaction chamber.
In a second aspect, the present disclosure provides a method of attaching a unique molecular identifier to a microcarrier.
Specifically, with the help of microchip technology, the micro-channels arranged in parallel are aligned to the micro-well reaction chamber array, different first molecular identifiers are respectively introduced into the channels, after the first molecular identifiers are connected with the micro-carriers, the unconnected identifiers are washed off, the micro-channels arranged in parallel are realigned to the micro-well reaction chamber array in the direction different from the direction of the channels, different second molecular identifiers are respectively introduced into the channels, the first molecular identifiers are combined with the second molecular identifiers, and the micro-carriers are connected with unique molecular identifiers through the modes of extension, amplification or connection and the like. Wherein the attaching of the unique molecular identifier to the microcarrier comprises attaching to the microcarrier of the present disclosure by any suitable method.
Wherein the first molecular identifier may be a nucleic acid sequence, i.e. a first nucleic acid molecular identifier, which comprises in the 5 'to 3' direction: a general domain, a first positioning domain and a connection domain. The second molecular identifier may be a nucleic acid sequence, i.e. a second nucleic acid molecular identifier, comprising in the 3 'to 5' direction: a connecting domain complementary region, a second positioning domain, a molecular marker and a capture domain precursor. The unique molecular identifier may be a nucleic acid sequence, i.e. a unique nucleic acid molecular identifier, comprising in the 5 'to 3' direction: a general domain, a first positioning domain, a connecting domain, a second positioning domain, a molecular marker and a capture domain. Wherein the unique nucleic acid molecule identifier comprises a nucleic acid sequence of the first nucleic acid molecule identifier that is complementary to the second nucleic acid molecule identifier and also comprises a nucleic acid sequence of the complementary nucleic acid sequence that has been extended, amplified, or ligated. By "unique" is meant that it is distinct from other nucleic acid molecule identifiers attached to microcarriers, nucleic acid sequences associated with cells or tissues, and nucleic acid sequences associated with the present disclosure.
Optionally, the species of molecular identifier comprises nucleic acid sequences, protein molecules, and other biomolecules.
Optionally, the number of the pore channels in the parallel arrangement micro-channels is 1 or more. For example, the parallel microchannels may comprise at least 10, at least 100, at least 1000, or at least 10000 microchannels arranged in parallel.
Optionally, the width of the pore channel in the parallel arrangement micro-channel is in the range of 0.1nm to 1000 μm. For example, the width of the parallel microchannels may be 1, 10, 100, 1000. mu.m.
Optionally, the width of the space between the channels in the parallel arrangement micro-channels is in the range of 0.1nm to 1000 μm. For example, the width of the channel pitch may be 1, 10, 100, 1000 μm.
Optionally, the liquid inlet arrangement of the parallel arrangement of microchannels comprises each channel connected to a separate liquid inlet, a plurality of channels connected to the same liquid inlet, and a channel connected to a plurality of liquid inlets.
Optionally, the liquid outlet arrangement of the parallel arrangement of microchannels comprises each channel connected to a separate liquid outlet, and also comprises a plurality of channels connected to the same liquid outlet or one channel connected to a plurality of liquid outlets.
Optionally, the method for linking the molecular identifier to the microcarrier comprises linking to the microcarrier by chemical immobilization, and also comprises promoting the completion of the linking reaction by thermal reaction or excitation light of a specific wavelength.
Optionally, the method of linking the microcarrier to the molecular identifier comprises a method of prior functionalization of the microcarrier, wherein the functionalization comprises a chemical, physical, biological means, such as activation of chemical groups within the microcarrier or incorporation of reactive functional groups into the microcarrier structure for said linking.
Optionally, the functional groups previously functionalized with the microcarriers comprise functional groups that are reactive or capable of being activated to form precursors with reactive functional groups, such as carboxylic acid groups activated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), aldehyde groups, epoxy groups, 1, 4-phenylisothiocyanate isocyanate (PDITC) groups, and the like.
Alternatively, the universal domain may comprise a functional group modification site comprising any reactive species capable of binding to a microcarrier and also a precursor capable of being activated to form a reactive functional group, and a PCR universal amplification start. Any suitable sequence may be used as the universal amplification start for PCR in accordance with the present invention. By "suitable sequence" is meant a sequence that does not interfere with the interaction between the tissue sample nucleic acid (e.g., RNA) and the capture domain, while the sequence can be complementarily bound to a universal primer for amplification of a nucleic acid molecule (e.g., cDNA).
Optionally, the length of the universal PCR amplification start sequence is at least 1 nucleotide, for example, the length can be 2, 10, 50, 100, 1000 nucleotides.
Alternatively, the universal domain may contain a cleavage domain for releasing the generated nucleic acid molecule identifier from the microcarrier, such as a poly-U oligonucleotide sequence.
Alternatively, the linking function of the molecular identifier to the microcarrier may be performed by its inherent chemical group or by introducing a group to the molecular identifier.
Optionally, the linking means of the molecular identifier to the microcarrier includes, but is not limited to, physical, chemical, biological modifications, such as electrostatic binding, amino modification, biotin group modification, phosphorylation modification, photocatalytic modification, radical polymerization, and the like.
Optionally, a washing step may be performed after the nucleic acid molecule identifier is attached to the microcarrier. The purpose of this step is to reduce non-specific adsorption of nucleic acid molecule identifiers and the microcarriers. This step may be performed using any method known in the art, and preferably, a buffer containing a surfactant, a salt, etc. may be used.
In the methods of the present disclosure, the first localization domain may also be defined as a first characteristic identification domain or a first tag domain, which may be regarded as a tag, a label or a name of the nucleic acid, and is generally located downstream of the universal domain, adjacent to the universal domain, and the first localization domain may be distinguished from each other, and may provide spatial position information for the nucleic acid sequence of the tissue or cell captured by the capture domain (e.g., determining the spatial position of the captured nucleic acid in the tissue or derived from a cell) without interfering with the capture of the nucleic acid sequence of the tissue or cell sample, and any suitable sequence may be used as the first localization domain of the present invention. By "suitable sequence" is meant that the sequence does not affect the interaction between the tissue sample nucleic acid (e.g., RNA) and the capture domain of the capture probe.
Alternatively, the different first molecular identifiers "different" means that the different first molecular identifiers are different from each other relative to other identifiers of the disclosure attached to microcarriers and other identifiers of the disclosure, and the different first molecular identifiers can provide spatial location information for omic information captured in a tissue or cell (e.g., determining the spatial location of a captured nucleic acid in a tissue or derived from a cell);
optionally, the first localization fields of the first molecular identifier that leads into each of the parallel arrangement microchannels are different and distinguishable from each other.
Optionally, the first localization domain sequence is at least 1 nucleotide in length, e.g., the length can be 2, 10, 50, 100, 1000 nucleotides.
In the methods of the present disclosure, the second localization domain may also be defined as a second characteristic identification domain or a second tag domain, which may be regarded as a tag, a label or a name of the nucleic acid, generally located downstream of the universal domain, adjacent to the universal domain, and which may be distinguished from each other, and which may provide spatial position information for the nucleic acid sequence of the tissue or cell captured by the capture domain (e.g., determining the spatial position of the captured nucleic acid in the tissue or derived from a cell) without interfering with the capture of the nucleic acid sequence of the tissue or cell sample, and any suitable sequence may be used as the second localization domain of the present invention. By "suitable sequence" is meant that the sequence does not affect the interaction between the tissue sample nucleic acid (e.g., RNA) and the capture domain of the capture probe.
Alternatively, the "different" of the different second molecular identifiers means that the different second molecular identifiers are different from each other relative to other identifiers of the disclosure attached to microcarriers and other identifiers of the disclosure, and the different second molecular identifiers can provide spatial location information for omic information captured in the tissue or cell (e.g., determining the spatial location of the captured nucleic acid in the tissue or derived from a cell);
optionally, the second domains of the second molecular identifier that lead into each of the parallel-arranged microchannels are different and distinguishable from each other.
Optionally, the second positional domain sequence is at least 1 nucleotide in length, e.g., the length can be 2, 10, 50, 100, 1000 nucleotides.
In the methods of the present disclosure, the linker may be any suitable nucleic acid sequence that is complementary to the linker complementary region according to the Watson-Crick base complementary pairing rules. The partial sequence does not affect the interaction between the tissue sample nucleic acid (e.g., RNA) and the capture domain of the capture probe and subsequent steps.
In the methods of the present disclosure, the linker complementary region can be any suitable nucleic acid sequence that is complementary to the linker according to the Watson-Crick base complementary pairing rules. The partial sequence does not affect the interaction between the tissue sample nucleic acid (e.g., RNA) and the capture domain of the capture probe and subsequent steps.
Optionally, the length of the sequence of the linker domain and the linker domain complement is at least 1 nucleotide, for example, the length may be 2, 10, 50, 100, 1000 nucleotides.
In the methods of the present disclosure, the molecular tags refer to nucleic acid sequences that provide information on the type of nucleic acid that hybridizes to the nucleic acid molecule identifier, and oligonucleotides conjugated to the same microcarrier may include different molecular tags. The sequence of the nucleic acid sequence of the molecular marker is unique. A molecular marker may also be defined as a Unique Molecular Identifier (UMI) that contains a type of nucleic acid (e.g., mRNA) used to distinguish hybridization to different nucleic acid molecular identifiers.
Alternatively, the molecular tag is at least 1 nucleotide in length, e.g., the length can be 2, 10, 50, 100, 1000 nucleotides.
In the methods of the present disclosure, the capture domain precursor may comprise a nucleic acid sequence for forming a capture domain. The capture domain precursor may comprise a nucleic acid sequence consisting of a poly-A sequence.
Optionally, the capture domain precursor is at least 1 nucleotide in length, e.g., the length can be 2, 10, 50, 100, 1000 nucleotides.
The method of the present disclosure comprises the step of incubating the first nucleic acid molecule identifier and the second nucleic acid molecule identifier after hybridization complementation with a reaction mixture to allow the microcarrier to generate the unique nucleic acid molecule identifier (fig. 4), wherein the unique nucleic acid molecule identifier comprises in the 5 'to 3' direction: a universal domain, a first localization domain, a linking domain, a second localization domain, a molecular tag, a capture domain, also including: the kit comprises a general domain, a first positioning domain, a second positioning domain, a molecular marker and a capture domain. The reaction mixture may contain any components that allow extension, amplification or ligation of a nucleic acid sequence, for example, nucleotides, an amplification enzyme (e.g., DNA polymerase) or a ligase (e.g., T4 DNA ligase), buffers, ultra pure water, and the like.
Optionally, the capture domain comprises a nucleic acid sequence that can capture a nucleic acid sequence.
Alternatively, the capture domain may comprise a random sequence, which may be used in combination with a poly-T oligonucleotide sequence (or poly-T analog, etc.) to facilitate capture of mRNA.
Alternatively, the capture domain may comprise a completely random sequence, and may be a degenerate capture domain according to principles known in the art.
Alternatively, the capture domain may comprise a poly-T oligonucleotide sequence that binds to a sequence complementary thereto, such as a nucleic acid (e.g., mRNA) carrying a poly-A sequence. The capture domain is not limited to a poly-T oligonucleotide sequence, and comprises a sequence functionally or structurally similar to a poly-T oligonucleotide sequence, such as a poly-U oligonucleotide or an oligonucleotide combined with deoxythymidine analogs or the like, wherein the oligonucleotide retains the functional properties of binding to a poly-A sequence.
Alternatively, the capture domain may be at least 1 nucleotide in sequence length, preferably at least 5, 10, 15, 20, 30 nucleotides in sequence length.
Alternatively, the capture domain may comprise a nucleic acid sequence capable of directing a reverse transcription reaction and may also comprise a nucleic acid sequence capable of generating a complementary nucleic acid molecule to the captured nucleic acid molecule.
Alternatively, in the methods of the present disclosure, the arrangement of the functional regions of the first nucleic acid molecule identifier, the second nucleic acid molecule identifier, and the unique nucleic acid molecule identifier includes, but is not limited to, the order, position, or content listed in the present disclosure, and one or more of the functional sequences in the above-mentioned molecule identifiers may be arranged in any suitable order or content.
Optionally, a prehybridization step may be included prior to introducing the reaction mixture, wherein the first nucleic acid molecule identifier and the second nucleic acid molecule identifier are complemented by the linker-linker complementary region according to the Watson-Crick base complementary pairing rules, which facilitates the generation of unique nucleic acid molecule identifiers.
Preferably, after the unique nucleic acid molecule identifier is generated, a washing step may be performed, which may wash the inside of the microwell reaction chamber except for the microcarriers and the unique nucleic acid molecule identifier attached to the microcarriers.
Alternatively, the unique nucleic acid molecule identifier may be characterized by any method known in the art, for example, using a fluorescently labeled tag sequence or sequencing analysis, or the like.
In a third aspect, the present disclosure provides a method for reducing omic information cross-contamination during the capture of tissue sample spatial omic information.
Specifically, a solid phase or liquid phase compound is introduced into the micro-reaction chamber array for storing the microcarrier, a tissue slice is attached to the surface of the micro-well array, a tissue sample is embedded in the micro-well or spread on the surface of the micro-well, the position information of the microcarrier represented by a specific unique nucleic acid molecule corresponds to the position of the tissue one by one, the tissue sample is imaged, and the surface of the micro-well is covered with a porous membrane for preventing cross contamination among tissue sample space omics information. Adding a tissue permeabilizing solution on the surface of the porous membrane, capturing the nucleic acid sequence of the domain-restricted tissue in the microwell by the microcarrier through the unique nucleic acid molecule identifier, and cleaning the surface; incubating the reaction mixed solution in a micro-well array, extending and synthesizing hybrid chains with captured omics information to enable the captured nucleic acid sequences and unique nucleic acid molecule identifiers to form complementary double-stranded nucleic acid sequences, and then amplifying and building a library of the double-stranded nucleic acid sequences; and (3) recovering the nucleic acid sequence, analyzing the recovered nucleic acid sequence, and then, according to the information of the first positioning domain and the second positioning domain, corresponding the analyzed omics information derived from the tissue or cell sample to the spatial position of the tissue sample according to the position information, thereby obtaining the spatial omics information of the tissue sample.
The methods described in this disclosure can be used for spatial transcriptomics studies of tissues.
Specifically, a solid phase or liquid phase compound is introduced into the micro-reaction chamber array for storing the microcarrier, a tissue slice is attached to the surface of the micro-well array, a tissue sample is embedded into the micro-well, the surface of the micro-well is covered with a porous membrane for preventing cross contamination among tissue sample space omics information, a tissue permeabilizing liquid is added to the surface of the porous membrane, and at the moment, the microcarrier captures mRNA of a limited domain tissue in the micro-well through a capture domain of a unique nucleic acid molecule identifier, and the surface is cleaned; incubating the reverse transcription reaction mixed solution in a micro-well array, extending and synthesizing hybrid chains with captured omics information to enable the captured mRNA and a unique nucleic acid molecule identifier to form cDNA, and then amplifying and establishing a library for the cDNA; and recovering the nucleic acid sequence, analyzing the recovered nucleic acid sequence, and then, according to the first positioning domain information and the second positioning domain information, corresponding the analyzed transcriptomic information from the tissue or cell sample to the spatial position of the tissue sample according to the position information, thereby obtaining the spatial transcription information of the tissue sample.
Alternatively, the embedding of the tissue sample in the microwell comprises introducing the tissue sample into the microwell by any external force or by virtue of the intrinsic properties of the reaction chamber of the microwell, for example, by mechanical, physical, or chemical induction.
Alternatively, the solid or liquid compound species comprises any compound capable of introducing a tissue section into a microwell, including polymers, monomers, mixtures, such as polyacrylamide, polyvinyl alcohol, polyethylene glycol, dry ice, water, paraffin.
Alternatively, the solid or liquid phase compound may be added in a volume greater than, less than, or equal to the volume of the microwell, preferably in a volume equal to the volume of the microwell.
Alternatively, the porous membrane type may include any porous membrane, such as a Polydimethylsiloxane (PDMS) porous membrane, a polyethylene porous membrane.
Optionally, the porous membrane has a pore size in the range of 0.1nm to 100mm, preferably a pore size sufficient to allow penetration of tissue permeabilizing fluids while confining nucleic acid sequences (e.g., mRNA) from tissue in the microwells to the microwells.
Alternatively, to facilitate analysis of the spatial location of the unique molecular identifier corresponding to the tissue sample, the tissue sample may be imaged using any method known in the art, such as light, dark field, confocal imaging, and the like. This step may be performed before or after the step of treating the tissue sample, e.g. before or after the cDNA generation step of the method.
Alternatively, the tissue sample may be labeled using any method known in the art to enable detection during imaging. Such as tissue staining, fluorescent labeling, and the like.
Alternatively, the permeabilizing fluid may comprise any fluid that releases nucleic acid, protein molecules from a cell or tissue sample, such as enzymes.
Alternatively, the scope of application of the method for reducing lateral diffusion in the present disclosure includes the capture of any nucleic acid, protein, polysaccharide molecule, preferably mRNA molecule, in a tissue sample, and the use of the method in any nucleic acid, protein, polysaccharide molecule in a cell, e.g., tRNA, rRNA, viral RNA.
Optionally, the disclosed methods comprise the step of performing subsequent experiments after the microcarriers are recovered from the microwell reaction chamber array after the tissue permeabilization step.
In the methods of the present disclosure, methods are included for extending the unique nucleic acid molecule identifier and the hybridized strand of captured nucleic acid into a complementary double-stranded nucleic acid sequence by any method known in the art. For example, a reverse transcription reaction. The double-stranded nucleic acid sequence generated by this method can be viewed as a copy of the captured component of the tissue sample, reflecting information contained in the tissue sample, such as transcriptome information.
Alternatively, the reaction mixture may comprise any component that allows amplification, extension or ligation of the captured nucleic acid sequence (e.g., mRNA), for example, a reverse transcription reaction mixture that allows reverse transcription of an oligonucleotide sequence into a double-stranded nucleic acid sequence (e.g., cDNA).
Optionally, the capture domain comprises a sequence that can trigger the production of its complementary strand by the captured nucleic acid, including but not limited to production downstream of the unique nucleic acid molecule identifier.
Alternatively, the method of the present disclosure comprises a step of removing the nucleic acid strand from which the captured nucleic acid is located after the complementary double-stranded nucleic acid sequence is generated, for example, mRNA, and the step can be performed by any method known in the art, for example, chemical, physical, biological methods, etc.
Alternatively, in the methods of the present disclosure, the tissue section may be removed after the complementary double stranded nucleic acid sequence is generated. This step may be performed using any method known in the art, such as enzymatic degradation.
Alternatively, in the methods of the present disclosure, amplification of the unique molecular identifier may also be performed prior to synthesis of the complementary double-stranded nucleic acid sequence.
The method of the present disclosure comprises the step of unwinding said complementary double-stranded nucleic acid sequence into an oligonucleotide sequence and subjecting the sequence to its complementary strand synthesis, which step is understood to be the generation of a second strand of the complementary double-stranded nucleic acid sequence for the purpose of generating sequence information of the nucleic acid captured by the unique nucleic acid molecule identifier.
Alternatively, random primers may be used in the reaction that generates the second strand, whereby nucleic acid fragments of random length will be produced, which nucleic acid products may correspond to the information of the captured sequence.
Alternatively, in the reaction to generate the second strand, a specific primer, e.g., a template switch primer, may be used, whereby a unique nucleic acid molecule identifier-corresponding fragment of full length will be produced.
Alternatively, a template conversion method may be used in the reaction to generate the second strand. Such as SMART technology as is known in the art. Preferably, this step can be performed in situ on the microcarrier according to the invention.
Optionally, the polymerase species used in the reaction to produce the second strand comprises any enzyme associated with nucleic acids, such as DNA polymerases, RNA polymerases, DNA ligases, restriction endonucleases, transcriptases, reverse transcriptases, and the like.
Alternatively, nucleic acid sequence amplification linkers may be introduced in the reaction that produces the second strand, and these sequences may contain sites for binding to polymerase chain reaction or other amplification, extension reaction primers.
Optionally, the method of the present disclosure comprises the step of recovering from the microcarrier the unique nucleic acid molecule identifier or a complementary double stranded nucleic acid sequence generated therefrom with the captured nucleic acid. This step can be accomplished by any method known in the art, such as enzymatic cleavage release or high temperature or salt methods, in order to disrupt the interaction between the nucleic acid and the microcarrier.
The method of the present disclosure comprises a step of increasing the amount of the second strand (e.g., cDNA), which may be performed on the microcarrier, or after recovering the unique nucleic acid molecule identifier with captured nucleic acid information or its complementary double-stranded nucleic acid sequence from the microcarrier. The number of complementary strands of nucleic acid that can be produced by this step should be available for subsequent steps, such as sequencing analysis.
Alternatively, the method of increasing the amount of the second strand (e.g., cDNA) can be accomplished using any method known in the art, such as polymerase chain reaction. Alternatively, the template for the polymerase chain reaction may be a complementary double-stranded nucleic acid sequence containing a unique nucleic acid molecule identifier, and the product of this reaction may also serve as a template for subsequent reactions.
The method of the present disclosure comprises a step of constructing a library of target sequence nucleic acids containing the captured sequence information, which may be performed by any method known in the art, and preferably, the nucleic acid library construction may be performed after introducing Illumina primer sequences into the second strand amplification products by polymerase chain reaction.
Alternatively, prior to the construction of the nucleic acid library, the target sequence may be fragmented, which facilitates subsequent pooling and sequencing analysis, which may be performed using any method known in the art, such as physical, chemical or biological methods, and the like.
Alternatively, prior to the establishment of the nucleic acid library, the target sequence may be subjected to appropriate processing steps, such as end-point repair, tailing, etc., which may facilitate the establishment of the nucleic acid library. This step can be performed using any method known in the art, such as enzymatic treatment.
Alternatively, the primer sequence introduced in the second strand production reaction may be cleaved prior to establishing the nucleic acid library, and this step may be performed using any method known in the art, such as enzymatic cleavage.
Alternatively, a fragment length screening step of the amplified product of the cDNA may be performed prior to the establishment of the nucleic acid library. This step can be performed using any method known in the art, such as nucleic acid sequence length analysis, and the like.
Alternatively, prior to the construction of the nucleic acid library, specific sequences, such as sequencing primer binding site sequences and the like, may be introduced into the target sequence, which may increase the accuracy of the analysis results of the nucleic acid library.
Alternatively, prior to the establishment of the nucleic acid library, appropriate DNA molecule purification methods may be employed to remove potentially introduced interferences, such as non-target nucleic acid sequences, nucleotides, salts, etc., which facilitates the reliability of the analysis results. This step may be performed using any method known in the art, such as magnetic bead separation.
Alternatively, when the target sequence containing the captured sequence information is amplified and pooled, the amplification and pooling method comprises any known nucleic acid amplification and pooling method, such as adding sequencing or amplification adaptors, adding amplification reaction mixtures, pooling and amplifying the target sequence.
The method of the present disclosure comprises an analysis step of the nucleic acid library. The target nucleic acid sequence may be analyzed using any method known in the art. Generally, such methods are sequence-specific methods, which may employ amplification reaction type sequence analysis methods using primers directed to the sequence being analyzed, for example. Alternatively, the amplification reaction may be a linear or non-linear reaction, such as a Polymerase Chain Reaction (PCR), an isothermal amplification reaction (e.g., RPA), and the like.
Alternatively, the analyzing step may comprise analysis of the unique molecular identifier, whereby the spatial localization of the analyzed sequence may be obtained.
Alternatively, each of the complementary double-stranded nucleic acid sequence and the second strand thereof can be used for analysis, and the first generation sequencing, the second generation sequencing, the third generation sequencing and other analysis processes can be adopted. Alternatively, the sequence analysis method of the present invention may be based on any means known in the art, e.g., IlluminaTMTechniques, pyrosequencing, and the like.
Optionally, the method of the present disclosure comprises the steps of recovering, pooling, and analyzing the unique nucleic acid molecule identifier or any nucleic acid sequence derived therefrom that is converted from the captured nucleic acid, from the microcarrier, either on the microcarrier or after recovering the unique nucleic acid molecule identifier with the captured nucleic acid information or its complementary double stranded nucleic acid sequence.
Optionally, prior to said sequence analysis, appropriate DNA molecule purification methods may be employed to remove interfering substances, e.g. non-target nucleic acid sequences, nucleotides, salts, which may be introduced into the sample, which may be advantageous to increase the reliability of the results. This step may be performed using any method known in the art, such as magnetic bead separation.
Alternatively, the tissue sample in the methods of the present disclosure may be a tissue sample of any organism or spatial structure of an organism, e.g., plant, animal, fungus.
Alternatively, the tissue sample in the methods of the present disclosure may be any type or kind of tissue sample, for example, dead or live tissue samples, fresh tissue may be used as the tissue sample of the present disclosure. Tissue samples of the present disclosure also include any treated or untreated tissue sample, such as fixed, unfixed, frozen, normothermic, paraffin tissue samples. In one embodiment of the invention, frozen tissue samples are used and tissue embedding is performed by OCT compounds that facilitate tissue structure retention and tissue sectioning while being compatible with subsequent procedures.
Optionally, the methods of the present disclosure comprise using the methods of the present disclosure to obtain or retrieve omics information unique to or independent of individual cells.
Optionally, the methods of the present disclosure comprise using the methods for omics analysis of any cell or any cell type in a sample, e.g., a blood sample. That is, the cells to which the methods of the present disclosure are applicable are not only tissue cells, but may also be single cells (e.g., cells isolated from non-fixed tissue). The single cell comprises a cell fixed at a position of the tissue and also comprises a single cell suspension introduced into the microwell.
Optionally, the methods of the present disclosure comprise using the methods for the capture and detection of omics information in any biological sample, e.g., for capturing DNA, mRNA, protein molecules, tRNA, rRNA, viral RNA in cells, tissues, viral samples.
Optionally, the methods of the present disclosure comprise the use of the methods of the present disclosure for any kind of biological omics testing and analysis, such as transcriptomics, genomics, epigenomics, proteomics, metabolomics.
Compared with the prior art, the invention has the following advantages:
1. the preparation process is simple, and the high-flux nucleic acid molecule identifier array with the space orientation domain can be prepared by microchip operation twice, so that the instrument cost for preparing the chip is effectively reduced.
2. The preparation method of the invention avoids the full-length synthesis of the capture probe, reduces the use variety of the required capture probe and effectively reduces the material cost required for preparing the space orientation domain.
3. The unique nucleic acid molecule identifier array prepared by the invention has higher resolution, the array line width range is 0.1nm-1000 mu m, and the single cell resolution can be achieved.
4. The unique nucleic acid molecule identifier array prepared by the invention has high modification density, and the integrity of obtaining the space omics information in the tissue sample is obviously improved.
5. The unique nucleic acid molecule identifier array prepared by the invention has uniform effect in the modification region, and ensures the uniformity of tissue sample space omics information acquisition.
6. The method effectively reduces the transverse diffusion of the space omics information in the tissue sample, and lays a foundation for the high-resolution space omics information capture.
7. The method finds extended application in genomics, epigenomics, proteomics, metabolomics, e.g. for mutation or epigenetic analysis of tissue cells.
Drawings
FIG. 1 is a diagram of a microwell array for dispersive microcarriers for use in a high resolution spatial omics detection method of tissue samples according to an embodiment of the present invention;
FIG. 2 is a diagram of an array of microwell reaction chambers for use in a high resolution spatial omics method of tissue sample detection according to an embodiment of the present invention;
FIG. 3 is a diagram of microchip channels for modifying nucleic acid molecule identifiers on microcarriers in a high resolution spatial omics detection method for tissue samples according to an embodiment of the present invention;
fig. 4 is a diagram of a microcarrier with a unique nucleic acid molecule identifier attached thereto, according to an embodiment of the present invention, for use in a high resolution spatial omics detection method of a tissue sample;
FIG. 5 is a diagram of a quality control of microbeads linked with unique nucleic acid molecule identifiers by fluorescently labeled poly-A probe pairs for use in a high resolution spatial omics detection method of tissue samples according to an embodiment of the present invention;
FIG. 6 is a diagram illustrating the overall concept of using an array of microwell reaction chambers loaded with microcarriers for high resolution spatial transcriptome analysis in a high resolution spatial omics detection method for tissue samples according to an embodiment of the present invention;
FIG. 7 is a graph showing the determination of the concentration of cDNA amplification products in a high resolution spatial omics detection method for tissue samples according to an embodiment of the present invention;
FIG. 8 is a quality control diagram of cDNA amplification products in a high resolution spatial omics detection method for tissue samples according to an embodiment of the present invention;
FIG. 9 is a graph of nucleic acid library concentration determination in a high resolution spatial omics detection method for tissue samples according to an embodiment of the present invention;
FIG. 10 is a quality control diagram of a nucleic acid library used in a high resolution spatial omics detection method of a tissue sample according to an embodiment of the present invention.
Fig. 11 is a diagram of analysis of a nucleic acid library for use in a high resolution spatial omics detection method of a tissue sample according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
Example 1: microwell slide preparation
Placing the glass plate with the uniform chromium film and the photoresist layer under a mask plate of a transparent array for exposure for 10s by an ultraviolet lamp, and then placing the substrate in a developing solution for soaking for 30s to obtain the surface of the patterned photoresist array slide with the chromium layer screwed; then placing the glass slide in a chromium etching solution for soaking for 5min, and then placing the glass slide in a glass etching solution (mass ratio HF: HNO)3：NH4F：H2O25: 23.5:9.35:450) for 80min to obtain a patterned microwell array slide (fig. 1), wherein the microwell height is 40 μm, the diameter is 30 μm, and the center-to-center spacing of the microwells is 60 μm.
Example 2: micro-reaction chamber preparation
Spreading the microsphere carrier suspension with the diameter of 5-7 μm on the surface of the microwell slide, standing for 5min, depositing the microspheres in the microwell array, cleaning the surface of the microwell slide, and washing off the microspheres outside the microwell array to obtain the microwell reaction chamber array containing the microspheres (figure 2).
Example 3: preparation of Microchip
Placing a glass plate with a uniform chromium film and a photoresist layer under a mask plate with pore channels arranged in parallel, exposing for 10s by an ultraviolet lamp, and then placing a substrate in a developing solution to soak for 30s to obtain the photoresist glass surface with a micro-pore channel pattern with a chromium layer; soaking in chromium etching solution for 5min, and placing the surface in glass etching solution (mass ratio HF: HNO)3：NH4F：H2Soaking in 25:23.5:9.35:450) for 40min to obtain a glass pore channel mold with the morphology structure of the micro-pore channel pattern; uniformly mixing Polydimethylsiloxane (PDMS) prepolymer and a curing agent according to a mass ratio of 10:1, vacuum degassing for 30min, pouring onto the surface of the glass mold, placing in an oven at 60 deg.C, curing for 10h, and lifting to obtain PDMS microfluidic channels (figure 3) arranged in parallel with a channel height of 10:120 μm, 40 μm in width and 60 μm in cell center-to-center spacing. Obviously, the preparation cost of the microchip is far lower than that of an ink-jet sample spotting machine, so that the instrument cost for modifying the unique nucleic acid molecule identifier is obviously reduced by adopting a microchip type modification method.
Example 4: preparation and characterization of beads with unique nucleic acid molecule identifiers attached thereto
Dispersing microbeads with the particle size of 5-7 mu m into a microwell array containing 10000 containable microcarriers to further form 10000 microwell reaction chambers, removing the microbeads outside the microwell array, and aligning 100 microchannels which are arranged in parallel to the microwell reaction chamber array. And respectively introducing first nucleic acid molecule identifier solutions into each pore channel, wherein the first nucleic acid molecule identifiers in the pore channels are different from each other (partial sequences are shown in table 1), so that the microbeads are connected with the first nucleic acid molecule identifiers.
Table 1: partial first nucleic acid molecule identifier sequence information
And introducing a washing solution into the pore channel, and washing away the unligated first nucleic acid molecule identifier. The 100 micro channels which are arranged in parallel are aligned to the micro-well reaction chamber array again in the direction vertical to the direction of the channels, second nucleic acid molecule identifiers are led into the channels, the second nucleic acid molecule identifiers among the channels are different from each other (partial sequences are shown in a table 2), the second nucleic acid molecule identifiers and the connecting domain of the first nucleic acid molecule identifiers generate hybridization complementation through the connecting domain complementation area, and then cleaning solution is led in to wash out the un-hybridized second nucleic acid molecule identifiers. In this embodiment, modification of 10000 different bead arrays of unique nucleic acid molecule identifiers is achieved by using 200 nucleic acid molecule identifiers, which significantly reduces the reagent cost required for full-length synthesis of sequences in the inkjet printing modification method in the prior art.
Passing an amplification reaction mixture through said microchannel and incubating at a constant temperature to generate said unique nucleic acid molecule identifier consisting of a 5 'to 3' end: a general domain, a first positioning domain, a connecting domain, a second positioning domain, a molecular marker and a capture domain. And (3) introducing a cleaning solution into the pore channel to clean the microbeads in the micro-well reaction chamber, so as to obtain the microbeads connected with the unique nucleic acid molecular identifiers (figure 4). And (2) dripping a fluorescence labeling nucleic acid sequence A20 (shown in table 3) which can generate hybridization complementation with the unique nucleic acid molecule identifier on the surface of the micro-well reaction chamber array, adopting a fluorescence microscope to characterize, wherein the modification result shows that the bead is connected with the unique nucleic acid molecule identifier (shown in figure 5), and carrying out amplification sequencing on the unique nucleic acid molecule identifier, and the result also shows that the unique nucleic acid molecule identifier has correct composition and contains the positioning domain, the molecular label and the capture domain. The modification resolution of the unique nucleic acid molecule identifier was 30 μm. In some embodiments, the unique nucleic acid molecule identifier is modified at a resolution significantly higher than existing ink jet printing modification methods. In addition, in this embodiment, the unique nucleic acid molecule identifier array is prepared at a high density and a spacing of 30 μm, which is lower than the prior art modification methods, and significantly improves the integrity of the acquisition of the spatial omics information in the tissue sample. In the embodiment, the unique nucleic acid molecule identifier array prepared by the invention has uniform modification effect, and the uniformity of tissue sample space omics information acquisition is ensured.
Table 2: partial second nucleic acid molecule identifier sequence information
Sequence name
Sequence information 5 '→ 3'
P2-1
NBAAAAAAAAAAAAAAAAAANNNNNNNNATATTGTGGCAGGCCAGT
P2-2
NBAAAAAAAAAAAAAAAAAANNNNNNNNCTAGGTGTGCAGGCCAGT
P2-3
NBAAAAAAAAAAAAAAAAAANNNNNNNNTTGCGTTCGCAGGCCAGT
P2-4
NBAAAAAAAAAAAAAAAAAANNNNNNNNGTACGACTGCAGGCCAGT
P2-5
NBAAAAAAAAAAAAAAAAAANNNNNNNNCTGTATTTGCAGGCCAGT
P2-6
NBAAAAAAAAAAAAAAAAAANNNNNNNNTCTGCGCCGCAGGCCAGT
P2-7
NBAAAAAAAAAAAAAAAAAANNNNNNNNCGATCATTGCAGGCCAGT
P2-8
NBAAAAAAAAAAAAAAAAAANNNNNNNNTTCTCTTGGCAGGCCAGT
P2-9
NBAAAAAAAAAAAAAAAAAANNNNNNNNTAGAGATCGCAGGCCAGT
P2-10
NBAAAAAAAAAAAAAAAAAANNNNNNNNCGCGTGTTGCAGGCCAGT
P2-11
NBAAAAAAAAAAAAAAAAAANNNNNNNNTAACTACCGCAGGCCAGT
P2-12
NBAAAAAAAAAAAAAAAAAANNNNNNNNCCCTCCTGGCAGGCCAGT
P2-13
NBAAAAAAAAAAAAAAAAAANNNNNNNNTAAGCGGAGCAGGCCAGT
P2-14
NBAAAAAAAAAAAAAAAAAANNNNNNNNTACTAGCAGCAGGCCAGT
P2-15
NBAAAAAAAAAAAAAAAAAANNNNNNNNATCGAAGTGCAGGCCAGT
P2-16
NBAAAAAAAAAAAAAAAAAANNNNNNNNTCGATACAGCAGGCCAGT
P2-17
NBAAAAAAAAAAAAAAAAAANNNNNNNNTAAGGAGCGCAGGCCAGT
P2-18
NBAAAAAAAAAAAAAAAAAANNNNNNNNTGTGTGCCGCAGGCCAGT
P2-19
NBAAAAAAAAAAAAAAAAAANNNNNNNNTGTCTGAGGCAGGCCAGT
P2-20
NBAAAAAAAAAAAAAAAAAANNNNNNNNCAACACGTGCAGGCCAGT
Example 5: tissue sample processing
Placing the container containing isopentane and the collection device in liquid nitrogen for precooling for 10min, then immersing the fresh mouse brain tissue in isopentane until the tissue is completely frozen, and then transferring to-80 ℃ for storage. Frozen mouse brain tissue was placed on pre-cooled OCT (polyethylene glycol and polyvinyl alcohol mixture) using a pre-cooled instrument, the exposed surface of the tissue was covered with OCT, and immediately after confirming that there were no air bubbles around the tissue, it was placed on dry ice until OCT was completely frozen. The tissue was cut to size and immediately frozen for sectioning (or transferred to-80 ℃ for sealed storage).
Precooling the microtome in advance, adjusting the cutting thickness to 10 mu m, then placing the microwell reaction chamber array in the microtome for precooling, placing the OCT frozen tissue sample on the microwell reaction chamber array, and cutting a tissue section with the thickness of 10 mu m.
Example 6: tissue sample staining
The microwell reaction chamber array was placed on a preheated PCR instrument with the sections facing up and incubated at 37 ℃ for 1 min. After fixing the tissue slices with pre-cooled methanol at-20 ℃ for 30min, removing methanol on the surface of the array, incubating the slice samples with isopropanol at room temperature for 1min and drying. And (3) dropwise adding hematoxylin staining solution to cover the section sample, incubating for more than 5min and less than 10min, discarding the staining solution and fully cleaning the microwell reaction chamber array. And (4) dropwise adding a blue-returning staining solution to cover the sliced sample, incubating for 2min, discarding the staining solution and fully cleaning the microwell reaction chamber array. An eosin staining solution (eosin: tris buffer (pH 6.0) ═ 1:9) was added dropwise to cover the sliced samples, incubated for 1min, the staining solution was discarded and the microwell reaction chamber array was washed thoroughly. Finally, the micro-well reaction chamber array is incubated at 37 ℃ for more than 5min, and the time is not more than 10 min. And (4) taking a picture by using a bright field microscope, and adjusting the exposure time and the shooting range to enable the boundary of the micro-well reaction chamber array to be visible.
Example 7: tissue permeabilization and cDNA Synthesis
Attaching a semipermeable membrane to the microwell reaction chamber array (fig. 6), dropping a tissue permeabilizing solution (0.1% pepsin, 0.1M hydrochloric acid) on the semipermeable membrane, incubating at 37 deg.C for 30min, removing the solution, adding sodium citrate buffer solution to the microwell reaction chamber array, washing, and removing the semipermeable membrane and waste solution. Adding reverse transcription reaction solution (containing hot-start reverse transcriptase and corresponding buffer system, template switching primer shown in Table 3) on the micro-well reaction chamber array, incubating at 50 deg.C for 1h, and removing the reverse transcription reaction solution.
A KOH solution (100mM) was added dropwise to the microwell reaction chamber array, and after incubation at room temperature for 3min, Tris buffer (10mM Tris-HCl, pH 8.5) was removed and added. The buffer was removed from the microwell reaction chamber array, the second strand synthesis reaction solution (containing Bst 2.0 polymerase and corresponding buffer system and corresponding primers, see table 3 for primer sequences) was added, the reaction solution was removed after incubation at 65 ℃ for 30min, and Tris buffer (10mM Tris-HCl, pH 8.5) was added. The buffer was removed again, KOH solution (100mM) was added, incubation was performed at room temperature for 5min, and then the solution was transferred to an EP tube without nuclease contamination, Tris buffer (1M Tris-HCl, pH 7.2) was added, and cryo-preserved.
Example 8: amplification of cDNA
Placing the mixture of the second strand synthesis product and the tris buffer solution on ice, adding a cDNA second strand amplification reaction solution (Taq enzyme, dNTP and a corresponding buffer system) and a primer (the sequence of the primer is shown in Table 3), fully mixing, and performing polymerase chain reaction, wherein the conditions are as follows:
step 1: heating at 98 deg.C for 3 min;
step 2: 15s at 98 ℃;
and step 3: 63 ℃ for 20 s;
and 4, step 4: 60s at 72 ℃;
and 5: go to step 2 for a total of 21 cycles;
step 6: 60s at 72 ℃;
and 7: and stopping at 4 ℃.
The product was stored at-20 ℃ until use.
The primer information is shown in Table 3.
Example 9: cDNA amplification product purification
Using a SPRISELECT nucleic acid fragment selection kit (Beckman Coulter), the amplification product was mixed with a SPRISELECT solution, allowed to stand at room temperature for 5 minutes or more, the magnetic beads were separated with a magnet, and the supernatant was discarded. Adding 80% ethanol solution into the magnetic beads, standing for 30s, separating the magnetic beads with a magnet, discarding the supernatant, repeating twice, and completely removing residual ethanol. Adding Tris buffer (10mM Tris-HCl, pH 8.5), pipetting, standing at room temperature for 2min, separating magnetic beads with magnet, collecting supernatant, and storing at low temperature. The cDNA fragment size distribution was determined using an automated electrophoresis system (Agilent technologies) and the cDNA concentration was determined using NanoDrop.
The concentration of the amplified cDNA and the purification results are shown in FIGS. 7 and 8. Carrying out reverse transcription on the hybrid sequence in the micro reaction chamber after capturing the mRNA, enriching and collecting the obtained cDNA, and then measuring the distribution and concentration of the cDNA, wherein the detection result shows that a cDNA sequence of 300-1000bp is generated and is consistent with the expected result.
Example 10: cDNA amplification product library construction
The purified product of cDNA was transferred to ice, and a pre-cooled Tris buffer (10mM Tris-HCl, pH 8.5) and a primer excision solution were added thereto, and the mixture was mixed well and transferred to a PCR instrument set at 4 ℃ in advance, followed by incubation at 32 ℃ for 5 min. Adding SPRISELECT solution into the reaction product, mixing well, standing at room temperature for 5min, separating magnetic beads with magnet, transferring the supernatant to a new EP tube, adding SPRISELECT solution, mixing well, standing at room temperature for 5min, separating magnetic beads with magnet, and discarding the supernatant. Adding 80% ethanol solution into the magnetic beads, standing for 30s, separating the magnetic beads with a magnet, discarding the supernatant, repeating twice, and completely removing residual ethanol. Tris buffer (10mM Tris-HCl, pH 8.5) was added thereto, and the mixture was pipetted and allowed to stand at room temperature for 2 min. Taking part of the supernatant, adding cDNA modification reaction solution (containing DNA ligase and corresponding buffer system and oligonucleotide adaptor shown in Table 3), mixing well, and incubating at 20 deg.C for 20 min. Mixing the reaction product with SPRISELECT solution, standing at room temperature for more than 5min, separating magnetic beads with a magnet, and discarding the supernatant. Adding 80% ethanol solution into the magnetic beads, standing for 30s, separating the magnetic beads with a magnet, discarding the supernatant, repeating twice, and completely removing residual ethanol. Adding Tris buffer (10mM Tris-HCl, pH 8.5), pipetting, standing at room temperature for 2min, separating magnetic beads with a magnet, pipetting a portion of the supernatant into a new EP tube, adding PCR reaction solution (Taq enzyme, dNTP and corresponding buffer system) and Illumina primer (table 3), mixing well, and performing polymerase chain reaction under the conditions:
step 1: at 98 ℃ for 1 min;
step 2: 20s at 98 ℃;
and step 3: 63 ℃ for 30 s;
and 4, step 4: 72 ℃ for 20 s;
and 5: go to step 2 for a total of 12 cycles;
step 6: 60s at 72 ℃;
and 7: and stopping at 4 ℃.
The nucleic acid library product was mixed with SPRISELect solution, allowed to stand at room temperature for more than 5min, the magnetic beads were separated with a magnet, and the supernatant was discarded. Adding 80% ethanol solution into the magnetic beads, standing for 30s, separating the magnetic beads with a magnet, discarding the supernatant, repeating twice, and completely removing residual ethanol. Adding Tris buffer (10mM Tris-HCl, pH 8.5), pipetting, standing at room temperature for 2min, separating magnetic beads with magnet, collecting supernatant, and storing at low temperature. The nucleic acid fragment size distribution was determined using an automated electrophoresis system (Agilent technologies) and the nucleic acid concentration was determined using NanoDrop. The concentration and distribution of the nucleic acid library after amplification are shown in FIGS. 9 and 10.
The information on the nucleic acid sequences is shown in Table 3, and the results of concentration and distribution of the amplified nucleic acid library are shown in FIGS. 9 and 10. The cDNA from the tissue and the unique nucleic acid molecule identifier in the micro-well reaction chamber is subjected to library construction, the sequence distribution after library construction is measured, and the detection result shows that the nucleic acid sequence with the length of 300-700bp is generated and is consistent with the expected result.
TABLE 3 example partial correlation sequence
Example 11: nucleic acid library analysis
Novaseq is selected to sequence the library, the sequenced nucleic acid library is subjected to sequence analysis, and the position information of the nucleic acid sequence is determined by combining the sequence information of the nucleic acid molecule identifier in the magnetic bead microcarrier. The target gene is matched to determine the expression information of the target gene at a specific position in space, and the molecular markers corresponding to the target gene are counted to count and quantify the expression of the target gene, so that the data error caused by the amplification deviation of the target gene can be reduced. The sequencing data is processed to match the unique nucleic acid molecule identifier array for each spatial point to a tissue staining image of the tissue section, thereby visualizing the sequencing data of the nucleic acid library at the spatial location of the tissue section.
The result shows that all corresponding nucleic acid molecule identifier information can be found in the analysis result of the nucleic acid library, the sequence information of the first positioning domain, the second positioning domain and the molecular marker is correct, the data quality can meet the analysis requirement, and the data analysis result shows that each micro-well reaction chamber can capture more than 2000 genes. Statistical data of nFature, nCount and mito are good, umap clustering results are correct, and typical gene staining results of Snap25, Slc32a1, Slc17a6, Aqp4 and Cldn5 brain tissues are correct (FIG. 11).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
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