CN114096678A - Multiple nucleic acid co-labeling support, and preparation method and application thereof - Google Patents

Multiple nucleic acid co-labeling support, and preparation method and application thereof Download PDF

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CN114096678A
CN114096678A CN202080005408.1A CN202080005408A CN114096678A CN 114096678 A CN114096678 A CN 114096678A CN 202080005408 A CN202080005408 A CN 202080005408A CN 114096678 A CN114096678 A CN 114096678A
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nucleic acid
sequence
cell
support
primer
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焦少灼
韩金桓
李研
刘书杰
马兴勇
罗云超
桑国芹
谢莹莹
徐猛
李宗文
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Beijing Xunyin Biological Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The invention provides a plurality of nucleic acid co-labeled supports and a manufacturing method and application thereof. The support comprises a support body and a plurality of nucleic acid markers located on the surface and/or inside the support body, wherein the nucleic acid markers on a single support at least comprise: one or more first nucleic acid labels, the function of which at least comprises capturing a specific compound in the reaction system to the surface of the support; one or more second nucleic acid labels, which function at least comprises a defined biochemical reaction process that can take part in a specific compound captured to the surface of the support. The support co-labeled by multiple nucleic acids can be used for 5 ' single cell RNA expression profiling analysis, 5 ' single cell VDJ library construction of a micropore array platform, 3 ' single cell RNA library construction, single cell transcriptome library construction, single cell multigroup research, multiplex PCR and/or multiplex PCR sequencing library construction and the like.

Description

Multiple nucleic acid co-labeling support, and preparation method and application thereof
Technical Field
The invention relates to a multi-nucleic acid co-labeling support and a manufacturing method and application thereof.
Background
Conventional nucleic acid molecule reactions, including nucleic acid hybridization, extension, amplification, etc., are performed in a liquid phase that provides a uniform and stable environment for the nucleic acid and enzyme reactions involved in the reactions to maximize yield. With the progress of biological research, scientists have found that attaching nucleic acids or enzymes involved in reactions to a solid phase surface can provide spatial position information to nucleic acids for easier purification, separation, detection and analysis, and thus have developed more and more solid phase nucleic acid reactions for nucleic acid sequence analysis and nucleic acid quantification, such as nucleic acid chip technology in which oligonucleotides are sequentially immobilized on a substrate, bridge amplification and microsphere emulsion amplification technology for second generation gene sequencing, nucleic acid-encoded microbeads for high throughput single cell sequencing, and the like.
Single Nucleotide Polymorphisms (SNPs) are widely used as genetic markers in population genomics, whole genome association studies (GWAS), paternity testing, and population identification. The traditional SNP identification technology can simultaneously analyze hundreds of sites, including TaqMan fluorescence analysis, KASPar identification technology and direct PCR sequencing technology, and has the advantages that the test operation is flexible when the number of the SNP sites to be detected is small, and the cost is increased linearly when the number of the SNP sites to be detected in a single sample is large (particularly more than or equal to 50); new detection techniques including biochips and high throughput sequencing can be implemented 103-106Identification of individual SNPs, such as RAD sequencing (restriction site associated DNA sequencing), exon sequencing, and whole genome sequencing, has the disadvantage of high individual sample detection pooling and sequencing costs, especially when the number of single sample SNPs is less than 1000, the average detection cost of a single SNP rapidly increases; when the number of the SNPs to be detected in a single sample is 20-1000, the average detection cost of the single SNP can be effectively reduced by using multiple PCR (polymerase chain reaction) libraries and combining second-generation sequencing.
The multiple PCR library construction generally refers to adding a plurality of pairs of PCR primers into a PCR amplification system to simultaneously amplify a plurality of target fragments, and then forming a nucleic acid library containing a joint and a sample tag required by a sequencer through a second step of universal primer amplification. Because a plurality of pairs of primers with higher concentration are added into a PCR amplification system, a large amount of primer dimers are easily formed in the multiplex PCR, and the quality of a constructed library is directly influenced by the primer dimers, so how to remove the primer dimers becomes a key link in the library construction of the multiplex PCR. It is reported that Zuiyi Yang et al can reduce primer dimer of multiplex PCR by optimizing the sequence of primer pair, and that RNA base modification of multiplex PCR primers by RNaseH-dependent PCR principle can also effectively reduce primer dimer during reaction. Although primer dimers can be removed by the methods disclosed above and by purification after PCR, multiplex PCR also has problems such as PCR bias due to differences in the efficiency of amplification of target regions and non-specific amplification due to primer pair cross-combinations. More importantly, because PCR amplification target regions of different primer pairs cannot overlap in a liquid phase condition, the continuous sequence analysis of single-reaction-tube long-fragment DNA is difficult to realize in a liquid phase system by common multiplex PCR.
As is well known, complex living organisms are composed of a plurality of cells with specific properties, and the types and the quantities of nucleic acids and proteins expressed by each cell are different under specific conditions, so that the detection of the nucleic acid and protein indexes on the single cell level is of great significance to the biomedical research. The single cell sequencing mainly comprises single cell genome sequencing, single cell RNA sequencing, single cell epigenome sequencing and space single cell sequencing from the detection index; from the detection flux, the method is mainly divided into low-throughput single-cell sequencing (detecting 1-500 cells at a time) and high-throughput single-cell sequencing (detecting 1000-10000 cells at a time).
The high-throughput single-cell RNA detection mainly comprises three realization modes, namely a water-in-oil-based droplet separation technology, a microplate-based beads labeling technology and microfluidics. Water-in-oil based droplet partitioning techniques are represented by the 10 Xgenomics, Drop-Seq and inDrop platforms. The technology wraps the barcode-marked micro-beads and single cells in oil drops through a microfluidic technology and cracks to release RNA containing polyA tail; each gel microbead is coupled with an oligo dT nucleic acid sequence containing a cell label and a molecular label; mRNA is bound to cell-tagged and molecularly tagged oligo dT nucleic acid molecules and then labeled with different cell tags by reverse transcription to cDNA from different cell sources and used for later library construction and sequencing analysis by mixing. The beads standard technology based on the micro-porous plate is represented by BD Cytoseq, Seqwell and microwell-seq. The technology naturally sinks cells to a micropore array with more than ten times of the number of the cells to ensure the single cell entry rate, and then adds microbeads marked by cell labels into micropores to capture mRNA after cell lysis; mRNA is bound to cell-tagged and molecularly tagged oligo dT, which is then reverse transcribed to different cell-derived cDNAs to label them with different cell tags and used for later pool building and sequencing analysis.
The drop method completely isolates cells and label microbeads from other cells and microbeads through water-in-oil, thereby effectively reducing the possibility of cross contamination; meanwhile, the liquid drop scheme can couple the cell label and the molecular label with the template switching sequence in addition to realizing a3 'RNA expression profile library, thereby realizing the sequencing of the 5' single-cell RNA expression profile; however, due to the instability and suspension characteristics of the liquid drops, the single cell library construction scheme based on the liquid drop method cannot change the liquid before and after the RNA is labeled by the cell label, so that the possibility of further carrying out complex reaction is reduced, and especially the position information of the labeled microbeads is lacked. The microplate method avoids the problem that the capture efficiency is influenced by probability collision in 10X, and has better cell capture efficiency; the label micro-beads have fixed positions after falling into the micropores, so that more liquid changing operations can be performed; however, all current microwell methods can only construct 3' single-cell RNA expression profiling libraries because microwells are semi-closed structures with open tops, which can cause cellular RNA to diffuse out of the wells.
Disclosure of Invention
It is an object of the present invention to provide a support co-labeled with a plurality of nucleic acids.
Another object of the present invention is to provide a method for preparing a support co-labeled with a plurality of nucleic acids.
Another object of the present invention is to provide the use of supports co-labeled with a plurality of nucleic acids.
The invention modifies more than two nucleic acid molecules on a support, wherein one nucleic acid molecule is used for capturing a target compound from a reaction pool and participates in a specific biochemical process together with other types of molecules which are marked on the surface of the same solid-phase compound, and the application directions comprise but are not limited to multiplex PCR library construction, single cell RNA expression profile, single cell transcriptome sequencing library construction, single cell multigroup sequencing library construction and the like.
In particular, in one aspect, the invention provides a support co-labeled with a plurality of nucleic acids, comprising a support body and a plurality of nucleic acid labels located on the surface and/or inside the support body, wherein the nucleic acids labeled on a single support comprise at least: one or more first nucleic acid labels, the function of which at least comprises capturing a specific compound in the reaction system to the surface of the support; one or more second nucleic acid labels, which function at least comprises a defined biochemical reaction process that can take part in a specific compound captured to the surface of the support.
According to a specific embodiment of the present invention, in the plurality of nucleic acid co-labeled supports of the present invention, the support bodies are solid beads and/or semi-solid hydrogel beads.
According to a particular embodiment of the invention, the plurality of nucleic acid co-labeled supports of the invention are compositions comprising a plurality of supports.
According to a specific embodiment of the present invention, in the supports co-labeled with multiple nucleic acids of the present invention, the number of the first nucleic acid label and the second nucleic acid label on the same support can be respectively greater than or equal to 1 and/or less than or equal to 1013And (4) respectively.
According to a specific embodiment of the present invention, in the multiple nucleic acid co-labeled support of the present invention, the sequences of the plurality of first nucleic acid labels on the same support are the same or different; the sequences of the first nucleic acid tags on different supports are the same or different; the sequences of the plurality of second nucleic acid tags on the same support may be the same or different; or the sequence of the second nucleic acid label on different supports may be the same or different.
In another aspect, the present invention provides a method for preparing a support co-labeled with a plurality of nucleic acids, comprising:
a plurality of nucleic acids are labeled onto the support body by grafting and/or grafting to obtain a plurality of nucleic acid co-labeled supports.
According to a specific embodiment of the present invention, the method for preparing a support co-labeled with a plurality of nucleic acids of the present invention comprises:
modifying the support body and the nucleic acid with functional units capable of interacting with each other, respectively, so that the two react to label the nucleic acid on the support body;
synthesizing nucleic acid directly on a support body according to a preset nucleotide sequence; and/or
Nucleic acid labeling is carried out on the support body using a protocol of biochemical reactions for nucleic acid extension or attachment.
On the other hand, the invention also provides application of the support co-labeled with multiple nucleic acids in 5 ' single cell RNA expression profiling, 5 ' single cell VDJ library construction of a micropore array platform, 3 ' single cell RNA library construction, single cell transcriptome library construction, single cell multigroup research, multiplex PCR and/or multiplex PCR sequencing library construction.
According to some embodiments of the invention, the multiple nucleic acid co-labeled supports of the invention are used for 5' single cell RNA expression profiling. Wherein: a template switching sequence containing a cell label and a molecular label and an RNA capture sequence are fixed on the support. In particular, the support is labeled with at least two nucleic acid sequences: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending or reverse transcribing as a primer; the second nucleic acid sequence comprises a cell tag sequence for tagging molecules derived from all mRNAs in the same cell; different types of supports have different cell tags on them. Preferably, the support is allowed to capture RNA released after single cell lysis in the microwells of the chip, and labeling of RNA derived from the same cell with the same cell tag is achieved by template conversion during reverse transcription, followed by amplification of cDNA and final construction into a library of 5' single cell RNA expression profiles.
According to some embodiments of the invention, the plurality of nucleic acid co-labeled supports of the invention are 5' single cell VDJ libraries used to construct microwell array platforms. Wherein: a template switching sequence containing a cell label and a molecular label and an RNA capture sequence are fixed on the support. In particular, the support is labeled with at least two nucleic acid sequences: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending or reverse transcribing as a primer; the second nucleic acid sequence comprises a cell tag sequence, a molecular tag sequence and a template switching sequence, wherein the cell tag sequence is used for marking molecules derived from all mRNA in the same cell; the molecular tag sequence is used for marking each reverse-transcribed cDNA molecule, and cDNA molecules reverse-transcribed from different RNAs on the same support are marked with different molecular tags; the template conversion sequence can be used as a template to continue extending the 3' end of the reverse transcribed cDNA so as to mark a molecular tag sequence and a cell tag sequence; different types of supports have different cell tags on them. Preferably, the support captures RNA released after single cell lysis in the micropores of the chip, and labeling RNA derived from the same cell with the same cell label is realized through template conversion in the reverse transcription process, and enrichment of TCR and BCR/Ig nucleic acid sequences is further realized through TCR and BCR/Ig gene constant region primers, and finally the construction of a high-throughput single-cell VDJ sequencing library is interrupted.
According to some embodiments of the invention, the multiple nucleic acid co-labeled supports of the invention are used to construct a 3' single cell RNA library. Wherein: the support has immobilized thereon conditionally-encapsivable random primers comprising a cell tag and an RNA capture sequence, and in particular, the support is labeled with at least two nucleic acid sequences: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending or reverse transcribing as a primer; the second nucleic acid sequence comprises a conditionally-closed random primer comprising a cell tag sequence for labeling molecules derived from all mRNAs in the same cell; different types of supports have different cell tags on them. Preferably, the support is allowed to capture RNA released after single cell lysis in the microwells of the chip and reverse transcribe into cDNA, subsequent random primers containing cell tags effect the same cell tag on cDNA derived from the same cell by two-strand synthesis, and cDNA amplification is then achieved by amplification to construct a 3' single cell RNA library.
According to some embodiments of the invention, the multiple nucleic acid co-labeled supports of the invention are used to construct a single cell transcriptome library. Wherein: random primer sequences containing cell labels and RNA capture sequences are fixed on the supports, different cell labels are arranged on different kinds of supports, and any sequence on the RNA molecule can be detected without being limited to the 3 'end or the 5' end; preferably, the support comprises two types of supports, each type of support having at least two nucleic acid sequences, a combination of a first nucleic acid sequence and a second nucleic acid sequence, or a combination of a third nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing a nucleic acid molecule of interest; the second nucleic acid sequence comprises a random primer sequence comprising a cell tag sequence for labeling molecules derived from all mRNAs in the same cell; the third nucleic acid sequence includes a cell tag sequence and a capture sequence. Preferably, the support is made to capture the RNA released after single cell lysis in the microwells of the chip and to label the same cell tags for RNA originating from the same cells during reverse transcription, followed by amplification of cDNA and final construction into a single cell RNA transcriptome library.
According to some embodiments of the invention, the multiple nucleic acid co-labeled supports of the invention are used for single cell multigenomic studies. Preferably, nucleic acid tags for constructing libraries of RNA expression levels and/or for detecting protein expression levels by proteins are included. Wherein: the support is immobilized with an RNA capture sequence comprising a cellular tag and a capture sequence of a nucleic acid tag for labeling the protein, and different types of supports have different cellular tags. Preferably, the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending as a primer; the second nucleic acid sequence comprises a cell tag sequence, a molecular tag sequence and a template switching sequence; cell tag sequences are used to label molecules derived from all mrnas in the same cell; the molecular tag sequence is used for marking each reverse-transcribed cDNA molecule, and cDNA molecules reverse-transcribed from different RNAs on the same support are marked with different molecular tags; the template conversion sequence can be used as a template to continue extending the 3' end of the reverse transcribed cDNA so as to mark a molecular tag sequence and a cell tag sequence; the third nucleic acid sequence comprises a cell tag sequence, a molecular tag sequence and a protein nucleic acid tag capture sequence, wherein the protein nucleic acid tag capture sequence is used for capturing and extending a protein nucleic acid marker which has the same spatial structure with the single cell to be detected. Preferably, the support captures the nucleic acid tags of RNA and protein released after single cell lysis in the micropores of the chip, and the same cell tags are marked on the RNA and protein nucleic acid tags from the same cell in the reverse transcription process, and then the RNA and protein nucleic acid tags are finally constructed into a single cell RNA transcriptome library and a protein marker nucleic acid library through amplification.
According to some embodiments of the invention, the multiple nucleic acid co-labeled supports of the invention are used to construct a multiplex PCR sequencing library. Wherein: primers capable of interfering with each other are immobilized on different supports, respectively. Specifically, the support comprises at least two types of supports: one or more first species of primer-labeled supports, one or more second species of primer-labeled supports, each support having at least one pair of nucleic acid primers labeled thereon: the first kind of primer-labeled support is labeled with a first nucleic acid primer pair, the second kind of primer-labeled support is labeled with a second nucleic acid primer pair different from the first nucleic acid primer pair, each of the two kinds of supports independently optionally comprises more nucleic acid primer pairs such as other nucleic acid primer pairs, and target fragments amplified by the primer pairs on the same support do not coincide on the template; the primer pairs labeled on different supports are different so that different regions of interest can be amplified, which may or may not be partially overlapping. Preferably, all the supports are mixed in proportion and then mixed with the nucleic acid template and the PCR enzyme reaction system, thereby performing single-tube unbiased multiplex PCR.
In another aspect, the invention also provides a kit comprising a plurality of nucleic acid co-labelled supports according to the invention. Preferably, the kit is a kit that can be applied to 5 ' single cell RNA expression profiling, 5 ' single cell VDJ library construction for microwell array platforms, 3 ' single cell RNA library construction, single cell transcriptome library construction, single cell multigenomic studies, multiplex PCR and/or multiplex PCR sequencing library construction.
More preferably, the kit further comprises one or more of the following compositions:
composition 1: a support mixture containing a template conversion sequence of a cell label and a molecular label and an RNA capture sequence, a microporous chip, a cell lysate, a reverse transcription reagent, a nucleic acid amplification reagent and a nucleic acid interruption library building module; the kit comprising the composition 1 can be used for 5' single cell RNA expression profiling;
composition 2: a support mixture containing a template conversion sequence of a cell label and a molecular label and an RNA capture sequence, a microporous chip, a cell lysate, a reverse transcription reagent, a constant region primer, a nucleic acid amplification reagent and a nucleic acid breaking library building module; the kit comprising the composition 2 can be used for constructing a 5' single cell VDJ library of a microwell array platform;
composition 3: a mixture of random primers containing cell tags and a support for RNA capture sequences, a microwell chip, a cell lysate, a reverse transcription reagent, a double-stranded synthesis module, and a nucleic acid amplification and extension reagent; the kit comprising the composition 3 can be used for constructing a 3' single-cell RNA library;
composition 4: a support mixture containing a random primer sequence of a cell label and an RNA capture sequence, a microporous chip, a cell lysate, a reverse transcription reagent, a double-strand synthesis module and a nucleic acid amplification and extension reagent; the kit containing the composition 4 can be used for constructing a single-cell transcriptome library;
composition 5: a capture sequence support mixture containing protein tag nucleic acid of a cell tag, a microporous chip, cell lysate, a reverse transcription reagent and a nucleic acid breaking library building module; the kit containing the composition 5 can be used for single-cell multigroup research;
composition 6: pre-mixing the primer-coupled support mixture with multiplex PCR enzymes and buffers; further optionally, the kit further comprises a tag primer adapted to the high-throughput sequencer; the kit containing the composition 6 can be used for multiplex PCR and/or construction of a multiplex PCR sequencing library (a premixed support mixture with coupled primers, multiplex PCR enzymes and a buffer solution can realize single-tube unbiased multiplex PCR; the kit further comprises a label primer adapted to a high-throughput sequencer, and the kit can be used for construction of the multiplex PCR library for sequencing analysis through index PCR).
In summary, the present invention provides a support co-labeled with multiple nucleic acids, and a method for preparing the same and applications thereof. The technology of the present invention can capture nucleic acid molecules to the surface of a solid phase and perform specific biochemical reactions together with other kinds of nucleic acids with the surface modified solid phase by performing various nucleic acid modification schemes on a solid phase (including semi-solid state) support. The specific type of multi-nucleic acid modified solid phase support can be used in the fields of multiple PCR library construction, single-molecule long-fragment nucleic acid sequencing library construction, single-cell transcriptome sequencing library construction, single-cell multibiology sequencing library construction and the like.
Drawings
FIGS. 1A to 1C are schematic structural views of a support co-labeled with a plurality of nucleic acids according to the present invention.
FIGS. 2A and 2B are schematic diagrams of the application of the supports co-labeled with various nucleic acids of the present invention to multiplex PCR reactions.
FIG. 2C is a schematic diagram of the use of various nucleic acid-tagged supports of the present invention in multiplex PCR sequencing library construction.
FIGS. 2D and 2E are schematic diagrams of the design method and structure of the multiple nucleic acid co-labeled supports for multiplex PCR according to the present invention.
FIG. 3A is a schematic diagram of the structure of a support co-labeled with various nucleic acids for 5 'single cell VDJ library application in 5' single cell RNA expression profiling and construction of microwell array platforms in accordance with the present invention.
FIG. 3B is a flow chart of an experiment in which the multi-nucleic acid-tagged support shown in FIG. 3A is applied to 5 'single-cell RNA expression profiling to construct a 5' single-cell VDJ library of a microwell array platform.
FIG. 3C is a schematic diagram of the preparation of a nucleic acid co-labeled support for 5' single cell RNA expression profiling according to the present invention.
FIG. 4A is a schematic diagram of the structure of a support co-labeled with a plurality of nucleic acids applied to a 3' single-cell RNA library according to the present invention.
FIG. 4B is a schematic diagram of the process of applying the various nucleic acid co-labeled supports of the present invention to a 3' single cell RNA library.
FIG. 5A is a schematic diagram of the structure of a plurality of nucleic acid co-labeled supports for constructing a single-cell transcriptome library according to the present invention.
FIG. 5B is a schematic diagram of a process for constructing a single-cell transcriptome library using a support co-labeled with a plurality of nucleic acids according to the present invention.
FIG. 6A is a schematic diagram of the structure of a plurality of nucleic acid co-labeled supports for constructing multi-genomic single-cell libraries according to the present invention.
FIG. 6B is a schematic diagram of a process for constructing a multi-component single-cell library using a multi-nucleic acid co-labeled support of the present invention.
FIG. 7A shows the results of agarose electrophoresis of the membrane protein nucleic acid tag sequencing library constructed using the procedure in example 1.
FIG. 7B shows the results of fragment analysis of the 5' single cell expression profiling library constructed using the procedure of example 1.
FIG. 7C shows the results of fragment analysis of a T cell VDJ library constructed using the procedure of example 1.
FIG. 7D shows the results of a fragment analysis of the B cell VDJ library constructed using the procedure of example 1.
FIG. 8 shows the distribution of reads at the gene level obtained by sequencing analysis using the 3' single cell RNA library constructed according to the procedure in example 2 and the single cell transcriptome library constructed according to the procedure in example 3. The BD Phapsody 3' single cell expression profiling library was a library analysis structure constructed completely using BD Rhapsody.
FIG. 9 shows the results of fragment size analysis of the multiplex amplification PCR sequencing library constructed using the procedure in example 4.
Detailed Description
The following detailed description and examples are intended to provide a better understanding of the nature and character of the present invention, and are not intended to limit the scope of the invention. The following detailed description and examples are given without specific reference to procedures which are conventional in the art or which are set forth by the manufacturer of the apparatus.
The use of the terms "a" and "an" and "the" in the description of the invention are intended to include the plural form as well, unless the context clearly indicates otherwise or the context clearly indicates otherwise.
The present invention provides, in a first aspect, a structure of a support co-labeled with a plurality of nucleic acids. As shown in FIGS. 1A to 1C, the support body may be a solid plane (FIG. 1A), a solid bead (FIG. 1B) or a semi-solid hydrogel (FIG. 1C); the nucleic acid markers can be located on a solid surface (FIGS. 1A and 1B) or within a loose interior of the hydrogel (FIG. 1C). The nucleic acids labeled on the individual supports include at least: one or more first nucleic acid labels 101, whose function at least comprises capturing a specific compound in the reaction system to the surface of the support (thus the first nucleic acid labels 101 are also referred to as capture nucleic acid labels); one or more second nucleic acid labels 102, which function at least comprises a defined biochemical reaction process (thus the second nucleic acid label 102 is also referred to as a reactive nucleic acid label) that can take part in a specific compound captured to the surface of the support. The support may optionally include other types of nucleic acid tags 1N.
According to some embodiments of the invention, a plurality of nucleic acid co-labeled supports of the invention are compositions comprising a plurality of the supports described above (supports of the structures illustrated in FIG. 1A, FIG. 1B, and/or FIG. 1C).
For certain applications, the sequences of the plurality of first nucleic acid tags 101 on the same support may be identical. For certain applications, the sequences of the first nucleic acid markers 101 may not be identical on the same support.
For particular applications, the sequence of the first nucleic acid markers 101 on different supports may be the same. For particular applications, the sequence of the first nucleic acid markers 101 on different supports may be different.
For particular applications, the sequences of the plurality of second nucleic acid tags 102 on the same support may be identical. For particular applications, the sequences of the plurality of second nucleic acid tags 102 on the same support may not be identical.
For particular applications, the sequence of the second nucleic acid labels 102 on different supports may be the same. For particular applications, the sequence of the second nucleic acid labels 102 on different supports may be different.
Similarly, for a particular application, the sequences of a plurality of other types of nucleic acid tags 1N on the same support may be the same or different. For particular applications, the sequences of other types of nucleic acid tags 1N on different supports may be the same or different.
The number of the first nucleic acid label, the second nucleic acid label and other types of nucleic acid labels on the same support can be respectively more than or equal to 1 and/or less than or equal to 1013And (4) respectively.
For a particular application, the functions of the first nucleic acid label and the second nucleic acid label can be switched, that is, the same nucleic acid label can have the functions of capturing a specific compound on the surface of the support in the reaction system and participating in a specific biochemical reaction process of the specific compound captured on the surface of the support. For example, such a first nucleic acid marker and a second nucleic acid marker can be both primers of a primer pair.
The invention also provides a method for preparing the support for co-labeling multiple nucleic acids for different purposes. Nucleic acid labeling of the support can be carried out using both the "graft to" and "graft from" protocols. For particular applications, the "gradient to" protocol may be used alone to label the support with nucleic acids. For particular applications, the "graff from" protocol may be employed solely for nucleic acid labeling of the support. For specific applications, nucleic acid labeling of the support may be combined with both "graft to" and "graft from" protocols.
When the "graft to" protocol is employed, the support and nucleic acid are each modified with functional units capable of interacting, including, but not limited to, one or more of hydroxyl, aldehyde, epoxy, amino, carboxyl and activated forms thereof, phosphate, alkynyl, azide, sulfhydryl, alkene, biotin, avidin, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, tosyl ester, and the like. The different types of nucleic acid tags (the first nucleic acid tag, the second nucleic acid tag, and the other types of nucleic acid tags) may be modified with the same functional unit or with different functional units. The modified support and nucleic acid are contacted under conditions sufficient to allow the functional units capable of interaction to react and become attached to each other.
When the "gradient from" protocol is used, the nucleic acid may be directly synthesized on the support according to a predetermined nucleotide sequence, or the nucleic acid may be labeled by a biochemical reaction for extending or ligating the nucleic acid. The labeled nucleic acids may be modified with nucleic acids well known in the art, including but not limited to one or more of amino, phosphate, alkynyl, azide, sulfhydryl, disulfide, alkene, biotin, azobenzene, methyl, spacer, photocleavable group, dI, dU, LNA, XNA, nucleobases, and dideoxynucleobases, among others.
The invention also provides the use of a plurality of nucleic acid co-labeled supports for multiplex PCR. In the conventional multiplex PCR application, all templates and primers are mixed in the same reaction system, and the types and total concentrations of primer pairs are increased correspondingly as the target region of PCR is increased, so that primer dimers are easily formed to reduce the amplification efficiency of the target region. The support co-labeled by multiple nucleic acids provided by the invention can well reduce the generation of primer dimers in multiplex PCR, and can realize single-tube analysis of the continuous sequence of a long fragment due to the avoidance of mutual interference among PCR primers. As shown in FIG. 2A, in this use, the multiple nucleic acid co-labeled supports provided by the present invention comprise at least two types of supports: one or more first species of primer-labeled supports 1, one or more second species of primer-labeled supports 2, each support having at least one pair of nucleic acid primers labeled thereon: as shown in the figure, a first nucleic acid Primer pair (a first forward Primer 201F and a first reverse Primer 201R) is labeled on a first kind of Primer-labeled support 1, a second nucleic acid Primer pair (a second forward Primer 202F and a second reverse Primer 202R) different from the first nucleic acid Primer pair is labeled on a second kind of Primer-labeled support 2, each of which independently optionally further includes more nucleic acid Primer pairs such as other nucleic acid Primer pairs (other forward primers 2NF and other reverse primers 2NR) and can reduce mutual interference between the pairs of primers on the same magnetic bead by PCR Primer design software such as Primer Premier optimized Primer sequences, target fragments amplified by the pairs of primers on the same support do not coincide on a template, the pairs of primers labeled on different supports can be different so that different target regions can be amplified, these regions of interest may or may not be partially coincident. Each primer labelled on the support comprises at least a H-region that can bind to and extend from the region of interest: a first forward primer H region 201FH, a first reverse primer H region 201RH, a second forward primer H region 202FH, a second forward primer H region 202RH, other forward primer H regions 2NFH, other reverse primer H regions 2 NRH. In a particular application, each primer labelled on the support also comprises at least the region of the universal nucleic acid sequence U: a first forward primer U region 201FU, a first reverse primer U region 201RU, a second forward primer U region 202FU, a second reverse primer U region 202RU, other forward primer U regions 2NFU, other reverse primer U regions 2 NRU. The sequence of the forward primer U region FU or reverse primer U region RU of the primers on all supports in a particular application may be identical: the first forward primer U region 201FU is the second forward primer U region 202FU is the other forward primer U region 2NFU, and the first reverse primer U region 201RU is the second reverse primer U region 202RU is the other reverse primer U region 2 NRU. The sequence of the forward primer U region FU or reverse primer U region RU of the primers on all supports may be non-uniform for a particular application. In the implementation of the multiplex PCR, supports labeled with different types of nucleic acid primers are mixed with the multiplex PCR reaction system according to a preset ratio, the preset ratio is determined according to the amplification efficiency of nucleic acids on different types of supports, and the preset ratio can be as low as 0.01 times of the average ratio of the different types of supports (for example, primers P1/P2/P3 are labeled on magnetic beads to form first type magnetic beads, and primers P4/P5/P6 are labeled on magnetic beads to form second type magnetic beads, where the ratio refers to the ratio of the number of the first type magnetic beads to the number of the second type magnetic beads when the magnetic beads are mixed), and can be as high as 100 times of the average ratio of the different types of supports. The multiplex PCR reaction system at least comprises a DNA template, DNA polymerase, dNTP, buffer solution with proper concentration and the like. As shown in fig. 2B, at the beginning of the multiplex PCR reaction, one of the primers (e.g., the first forward primer 201F or the second forward primer 202F) labeled on the support will bind to the single-stranded DNA template 203, 204 in the reaction system to confine it to the surface of the support, and generate the complementary strand 205, 206 of the DNA template under the action of the polymerase, and then the complementary strand 205, 206 is dissociated from the template and bound by the other primer (e.g., the first reverse primer 201R, the second reverse primer 202R) on the same support and extended to obtain the nucleic acid strand 207, 208 complementary to the complementary strand 205, 206 of the DNA template; this is repeated to obtain a support with a large number of nucleic acid sequences. Because the primer pairs marked on the same support are limited in types and are physically far away from the primer pairs on other supports, the possibility of forming dimers by mutually combining different primer pairs can be effectively reduced; meanwhile, the target amplification region of the multiplex PCR can be effectively increased by labeling more supports with different kinds of nucleic acids and increasing the number of labeled primer pairs on each support, for example, less than 5 primer pairs can be coupled to each support, or less than 10 primer pairs can be coupled to each support, or less than 100 primer pairs can be coupled to each support.
Still further, the present invention provides the use of a plurality of nucleic acid co-labeled supports for multiplex PCR sequencing library construction. As shown in FIG. 2C, the support with the nucleic acid sequences 207, 208 complementary to the complementary strand of the DNA template generated in the reaction shown in FIG. 2B is used as a template for sequencer-compatible primer sequences: the third forward primer 209F and the third reverse primer 209R, and the second PCR reaction, thereby obtaining a nucleic acid sequencing library that can be used for sequencing. The sequencer-compatible primer sequences include at least the universal binding sequence (universal nucleic acid sequence U region, i.e. other forward primer U region 2 NFU/other reverse primer U region 2NRU) in the primers shown in fig. 2A, the sample tag 2NFi/2NRi and the sequencer-compatible nucleic acid sequence 2NFA/2NRA, where the forward universal nucleic acid sequence U regions are identical on all supports: the first forward primer U region 201FU ═ the second forward primer U region 202FU ═ the other forward primer U region 2NFU, and the reverse universal nucleic acid sequence U regions on all supports were also identical: the first reverse primer U region 201RU ═ the second reverse primer U region 202RU ═ the other reverse primer U region 2 NRU. Sequencers for sequencing include, but are not limited to, MGIseq sequencing platform, illumina sequencing platform, Ion sequencing platform, PacBio sequencing platform, Nanopore sequencing platform, and the like.
The invention also provides a method for preparing the multiple nucleic acid co-labeling supports for multiple PCR applications. As shown in fig. 2D, when sequencing analysis of the continuous base sequence of the long fragment 210 is required, single primer pair amplification is not sufficient, and at this time, multiple primer pairs are required to be designed for amplification and then constructed for library sequencing, such as amplifying the first target fragment 201 by using the first primer pair (represented by the H region of the primers in the scheme shown in fig. 2D, i.e., the first forward primer H region 201FH and the first reverse primer H region 201RH), amplifying the second target fragment 202 by using the second primer pair (the second forward primer H region 202FH and the second reverse primer H region 202RH), and amplifying more target fragments 2N by using more primer pairs (other forward primer H regions 2NFH and other reverse primer H regions 2NRH), and finally splicing the sequencing results into the sequence of the long fragment 210. In the conventional liquid phase multiplex PCR reaction, because the first target segment 201 is partially overlapped with the second target segment 202, the second forward primer and the first reverse primer generate small non-target amplification products together, the primers need to divide the multiplex PCR reaction into at least two tubes for parallel amplification, and the amplification target segments are not overlapped by primer pairs: the first forward primer, the first reverse primer, the other forward primers and the other reverse primers are one tube, and the second forward primer and the second reverse primer are one tube. When the multiplex PCR library construction method using a support labeled with multiple nucleic acids according to the present invention is used, a support co-labeled with multiple nucleic acids of a first kind can be obtained by labeling a first primer pair not overlapping with a target fragment for amplification and other primer pairs on the same magnetic bead, for example, a first magnetic bead, and a support co-labeled with multiple nucleic acids of a second kind can be obtained by labeling a second primer pair overlapping with the target fragment for amplification of the primer pair on the first magnetic bead on another magnetic bead, for example, a second magnetic bead, so that the primer pairs on the supports co-labeled with multiple nucleic acids of the two kinds do not interfere with each other even when amplification primers are carried out in the same tube. According to the principle of primer design shown in FIG. 2D, the present invention synthesizes beforehand the first primer pair 201F, 201R having the 5 ' specific modification 211, the second primer pair 202F, 202R having the 5 ' specific modification 211, and further other primer pair 2NF and 2NR having the 5 ' specific modification 211 (FIG. 2E). Specific 5' modifications include, but are not limited to, hydroxyl, aldehyde, epoxy, amino, carboxyl and activated forms thereof, phosphate, alkynyl, azide, sulfhydryl, alkene, biotin, avidin, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, tosyl ester and the like, and corresponding selected supports include, but are not limited to, functional groups 212 such as epoxy, amino, carboxyl, alkynyl, azide, alkene, heavy metal, azide, avidin and the like. Contacting and coupling a support with a functional group 212 with a nucleic acid primer with a5 'specific modification 211 under suitable conditions, in particular separately coupling primers capable of generating non-specific products to different supports, for example coupling a first primer pair 201F, 201R with a 5' specific modification 211, a further primer pair 2NF, 2NR with a5 'specific modification 211 to a first microbead to form a first product 213, coupling a second primer pair 202F and 202R with a 5' specific modification 211 to a second microbead to form a second product 214 (FIG. 2E), and finally mixing the first product 213 and the second product 214 together in a ratio to form the final support with multiple nucleic acid labels for multiplex PCR pooling.
The invention also provides the use of a plurality of nucleic acid co-labeled supports for 5' single cell RNA expression profiling. It is known that in a complex living organism, which is composed of many cells having specific properties, the species and amount of RNA to be expressed and transcribed in each cell are different under specific conditions, and therefore, it is important to detect RNA transcription at the single cell level. The current technology for detecting single cell transcriptome can be divided into medium-low flux and high-flux single cell transcriptome sequencing technology according to flux. The sequencing of the medium-low flux single-cell transcriptome takes smart-seq as a representative, and the single-cell transcriptome is constructed by the reverse transcription and amplification of RNA obtained by directly cracking single cells; library preparation of high-throughput single-cell transcriptome sequencing is represented by water-in-oil microfluidics and microwell array platforms, mRNA molecules derived from different cells are reverse-transcribed into cDNA molecules containing corresponding unique tags by oligo dT primers or Template Switch Oligonucleotides (TSOs) containing cell tags and molecular tags, and thousands of single-cell mRNA expression conditions can be simultaneously analyzed by further sequencing. The water-in-oil technology platform wraps a single cell and a single microbead containing a cell label in a single droplet to perform lysis reverse transcription one-step operation, and 3 'end or 5' end near RNA can be divided into a3 'single-cell RNA expression profile library and a 5' single-cell RNA expression profile library according to sequencing reading. The microwell array platform is usually an array chip consisting of 20-60 μ M diameter microwells, in which single cells are lysed and mRNA with polyA tail is captured by microbeads carrying cell-tagged oligo dT primers in the same well, and then RNA reverse transcription products derived from the same cells are labeled with the same and unique cell tags by reverse transcription extension. The efficiency of the current single cell sequencing library preparation platform based on the micropore array is greatly dependent on the RNA capture efficiency of oligo dT microbeads in micropores, and the micropore array platform different from a oil-in-water platform can only construct a 3' single cell RNA expression profile library. The invention can improve the RNA capture efficiency of the microbeads in the micropores by labeling the microbeads in the preparation of the single cell sequencing library with various nucleic acids, and can realize the preparation of the 5' single cell RNA expression profile library.
The invention provides application of a support co-labeled by multiple nucleic acids in 5 'single cell RNA expression profiling and construction of a 5' single cell VDJ library of a micropore array platform.
As shown in fig. 3A, the support is a microbead (solid microbead or semi-solid hydrogel microbead), and at least two nucleic acid sequences are labeled on the support: a first nucleic acid sequence 301 and a second nucleic acid sequence 304.
The first nucleic acid sequence 301 comprises at least a capture sequence 303 for capturing a nucleic acid molecule of interest and for extending or reverse transcription as a primer, for example the base sequence oligo dT with a length of 15-40, the efficiency of capturing RNA can be controlled by adjusting the number and density of the first nucleic acid sequence 301 on the support. The first nucleic acid sequence 301 also includes a first universal sequence nucleic acid 302 and a conditional break site X for a particular application. Conditional cleavable sites include, but are not limited to, one or more of disulfide modifications, dU modifications, RNA base modifications, dI modifications, DSpacer modifications, AP site modifications, photocleavable PC linkers, and restriction enzyme recognition sequences.
The second nucleic acid sequence 304 is composed of one or more of a second universal nucleic acid sequence 305, a cell tag sequence 306, a molecular tag sequence 307, and a template switch sequence 308. Wherein the second universal nucleic acid sequence 305 can comprise a linker nucleic acid sequence that matches a sequencer, such as Read1 Sequencing Primer or Read2Sequencing Primer in an illumina sequencer. Cell tag sequence 306 is used to label molecules derived from all mRNAs in the same cell, with the same cell tag on each support and different cell tags on different types of supports. The cell tag sequence 306 may be a random or semi-random nucleic acid sequence, such as 12bp degenerate NNNNNNNNNNNN, or may be a combination comprising a plurality of fixed nucleic acid sequences, such as 96 8-base sequences and 96 8-base sequences, with or without linking nucleic acid regions between the 8-base sequences. Molecular tag sequence 307 is used to label each reverse transcribed cDNA molecule, and cDNA molecules reverse transcribed from different RNAs on the same support are labeled with different molecular tags. Molecular tag 307 may be a random or semi-random nucleic acid sequence of 8-20 bases in length, such as 9 random degenerate bases NNNNNNNNN or NNNNNNNNNV. The template switch sequence 308 may serve as a template to extend the 3' end of the cDNA reverse transcribed from the first nucleic acid sequence 301 to tag the molecular tag sequence 307, the cell tag sequence 306, and the second universal nucleic acid sequence 305. The template switch sequence 308 includes more than two RNA bases rG or other modified base G analogs, such as LNA or XNA, at least at the 3' end.
FIG. 3B is a flow chart of the experiment of constructing a 5' single cell library of a microwell array platform using the multi-nucleic acid-labeled supports shown in FIG. 3A, wherein two nucleic acid labels are coupled to each support: a first nucleic acid sequence 301 and a second nucleic acid sequence 304. When a single support labeled with a first nucleic acid sequence 301 and a second nucleic acid sequence 304 is contacted with RNA derived from a single cell, RNA309 containing a sequence complementary to the second nucleic acid sequence 304 is captured by the first nucleic acid sequence 301 on the support and forms a cDNA molecule 310 by a reverse transcription reaction system, wherein the cDNA is extended to the 5' end of the RNA309 by a reverse transcriptase having a terminal nucleotidyl transferase function to add consecutive bases C to the cDNA strand, which then complementarily binds to the second nucleic acid sequence 304 containing two or more bases rG or a base analog thereof near the surface of the same support and continues to the region of the second universal nucleic acid sequence 305 to form a complete cDNA molecule 310 having a cell tag and a molecular tag. Alternatively, the cDNA molecules 310 may be detached from the support via the cleavable sites X as a template for the next amplification step, or an extended strand complementary to the cDNA molecules 310 formed by single primer extension of the second universal nucleic acid sequence 305 may be used as a template for the next amplification step, or a support containing the cDNA molecules 310 may be enzymatically treated to remove the first nucleic acid sequence 301 and the second nucleic acid sequence 304 that are not involved in the reverse transcription reaction and then used as a template for the next amplification step. In a subsequent step, the fragmented or unbroken cDNA molecules 310 are PCR amplified as templates with primer pairs containing a first universal sequence 302 and a second universal sequence 305 to form a double stranded nucleic acid 311 product. Further, the double-stranded nucleic acid 311 can be analyzed for the species and abundance of RNA expression of a single cell by two pooling schemes, one for unbiased analysis of the expression of all RNA molecules with polyA tail, in which the double-stranded nucleic acid 311 is randomly broken, end-repaired, and base A is added at the 3' end to form a molecular structure 312, and then ligated to a linker 313 containing an overhanging T and amplified by a first primer 315 containing a first sample Index317 and a second primer 319 containing a second sample Index321 to form a first final library 323. Wherein the first primer 315 comprises a first nucleic acid sequence 316 compatible with a sequencer, a first sample index317, and a sequence 318 complementary to the long-chain portion sequence in the linker 313. The second primer 319 comprises a second nucleic acid sequence 320 compatible with the sequencer, a second sample index321 and a sequence 322 identical to a partial sequence of the second universal nucleic acid sequence 305. Such a pooling method may be replaced by other random library construction schemes that serve the same purpose, including but not limited to, transposase-interrupted pooling or random primer extension pooling. Another library construction scheme for the targeted analysis of the expression of the target gene can be achieved by two-step multiplex PCR, such as first step multiplex PCR 325 and second step multiplex PCR 327 using the first gene-specific primer 324, second gene-specific primer 326 and the universal primer 305, respectively, and finally amplifying the second step multiplex PCR 327 as a template by the first primer 315 containing the first sample Index317 and the second primer 319 containing the second sample Index321 to form a second final library 328. The library constructed by targeted multiplex PCR can be used for analysis of the immune repertoire, and further, the second multiplex PCR product 327 can be used to construct a full-length VDJ immune repertoire library for analysis of T cell receptor and antibody VDJ sequences according to the first random break library construction scheme. Both the first final library 323 and the second final library 328 are further used for sequencing and information analysis.
The invention also provides a method for preparing the nucleic acid co-labeling support applied to the 5' single cell RNA expression profiling analysis. As shown in FIG. 3C, cell tag sequence 306 consists of 6 regions of first cell tag 329, first attachment region 330, second cell tag 331, second attachment region 332, third attachment region 333, and third cell tag 334 joined in series. To co-tag the first nucleic acid sequence 301 and the second nucleic acid sequence 304 on a support, the present invention synthesizes in advance the first nucleic acid sequence 301 and the third nucleic acid sequence 335 with a 5' specific modification 340. Specific 5' modifications 340 include, but are not limited to, hydroxyl, aldehyde, epoxy, amino, carboxyl and activated forms thereof, phosphate, alkynyl, azide, sulfhydryl, alkene, biotin, avidin, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, tosyl ester, etc., and the corresponding selected support includes, but is not limited to, functional groups 339 such as epoxy, amino, carboxyl, alkynyl, azide, alkene, heavy metal, azide, avidin, etc. Contacting and coupling (step 341) the support having functional group 339 with first nucleic acid sequence 301 and third nucleic acid sequence 335 having 5' modifications under suitable conditions to form first product 342, the ratio of first nucleic acid sequence 301 to third nucleic acid sequence 355 on first product 342 being adjustable by adding different concentrations; then the first product 342 is hybridized and extended with a fourth nucleic acid sequence 336 in a buffer environment containing appropriate salt ion concentration, dNTP and polymerase through a hybridization step 343 to obtain a second product 344, and the fourth nucleic acid sequence 336 nucleic acid molecule sequentially contains a complementary sequence 332 'of the second connecting region 332, a complementary sequence 331' of the second cell tag 331 and a complementary sequence 330 'of the first connecting region 330 from 5'; the third nucleic acid sequence 335 on the support hybridizes through its own linker region 330 sequence to the complementary sequence 330' on the fourth nucleic acid sequence 336 and extends to the sequence second cell tag 331 and second linker region 332; the second product 344 is ligated to the fifth nucleic acid sequence 346 under the action of DNA ligase after removal of the complementary strand under denaturing conditions to produce a third product 347; the fifth nucleic acid sequence 346 is double-stranded DNA resulting from pre-annealing hybridization of the first nucleic acid molecule 337 and the second nucleic acid molecule 338, the first nucleic acid molecule 337 comprising the third linker region 333, the third cellular tag 334, the molecular tag sequence 307, and the template switch sequence 308, which are linked in sequence, and the second nucleic acid molecule 338 comprising at least the complement of the partial sequences of the second linker region 332 and the third linker region 333, and under certain conditions also comprising the complement of the partial third cellular tag 334, such that the second nucleic acid molecule 338 can hybridize to the third linker region 333 on the first nucleic acid molecule 337 and the second linker region 332 on the second product 344 and be linked to each other by a ligase; in particular, the 5' end of the first nucleic acid molecule 337 sometimes also contains a phosphate modification; finally, the third product 347 is eluted through an elution step 348 under denaturing conditions to wash away the complementary nucleic acid sequence, i.e., the second nucleic acid molecule 338, to form a support 349 eventually labeled with the first nucleic acid sequence 301 and the second nucleic acid sequence 304, which support 349 labeled with the first nucleic acid sequence 301 and the second nucleic acid sequence 304 can be directly used in a pooling procedure for 5' single cell RNA expression profiling.
The invention also provides the application of the support co-labeled by multiple nucleic acids in the 3' single-cell RNA library, wherein the cell tag and the reverse transcription primer oligo dT are respectively positioned at two ends of the cDNA molecule. As shown in fig. 4A, at least two nucleic acid-labeled supports are shown, where the supports are microbeads (solid microbeads or semi-solid hydrogel microbeads) and the supports are labeled with at least two nucleic acid sequences: a first nucleic acid sequence 401 and a second nucleic acid sequence 404.
The first nucleic acid sequence 401 comprises at least a capture sequence 403 for capturing a nucleic acid molecule of interest and for extending or reverse transcription as a primer, such as the base sequence oligo dT with a length of 15-40, the efficiency of capturing RNA can be controlled by adjusting the number and density of the first nucleic acid sequence 401 on the support; the first nucleic acid sequence 401 may also include a first universal sequence nucleic acid 402 for a particular application.
The second nucleic acid sequence 404 is composed of one or more of a second universal nucleic acid sequence 405, a cell tag sequence 406, a primer sequence 407, and a reversible blocking site 408. Wherein the second universal nucleic acid sequence 405 can comprise a linker nucleic acid sequence compatible with a sequencer, such as Read1 Sequencing Primer or Read2Sequencing Primer of an illumina sequencer, and optionally the second universal nucleic acid sequence 405 can comprise a conditional break site X. Conditional cleavable sites include, but are not limited to, disulfide modifications, dU modifications, RNA base modifications, dI modifications, DSpacer modifications, AP site modifications, photocleavable PC linker and restriction enzyme recognition sequences. Cell tag sequence 406 is used to label molecules derived from all mRNAs in the same cell, with the same cell tag on each support and different cell tags on different types of supports. The cell tag sequence 406 may be a random or semi-random nucleic acid sequence, such as 12bp degenerate NNNNNNNNNNNN, or may be a combination comprising a plurality of fixed nucleic acid sequences, such as 96 8-base sequences and 96 8-base sequences, with or without linking nucleic acid regions between the 8-base sequences. The primer sequence 407 can be used as a primer to extend a cDNA molecule bound to the complementary primer, and can be bound to and extended from a cDNA product obtained by reverse transcription of the capture sequence 403; primer sequence 407 can be a random or semi-random nucleic acid sequence of 5-15 bases in length, such as 6 random degenerate bases NNNNNN, or can be used with gene-specific sequences to enrich for the targeted region. The reversible blocking site 408 functions to prevent non-specific extension of the primer sequence 407 as a primer when the target nucleic acid is captured and extended by the first nucleic acid sequence 401, and to release the blocking function under specific conditions to allow extension of the primer sequence 407 as a primer. Reversible blocking site 408 can be a simple 3 'phosphate modification, ddNTP modification or C3spacer modification, or can be a combination of cleavable modification and extension blocking modification, cleavable modification can be DSpacer modification/RNA base modification/dU modification, etc., extension blocking modification includes but is not limited to LNA/XNA/3' phosphate/inverted dT/ddNTP/C3 spacer/C6 spacer/various fluorescent dyes and quenching modification, etc., for example reversible blocking site 408 can be (rN) NN-C3 or (rN) N-C3-C3-ddN, rN represents any one ribonucleotide degenerate base, N represents any one deoxyribonucleotide base, C3 is extension blocking modification C3spacer, ddN is double deoxyribonucleotide degenerate; this sequence, after forming a double strand with the target DNA, can be cleaved by RNaseH recognition and exposes the 3' hydroxyl group of primer sequence 407, thereby activating the nucleic acid extension ability of primer sequence 407 as a primer.
FIG. 4B is a flowchart of the experiment for constructing 3' single-cell RNA library using the double-nucleic-acid-labeled support shown in FIG. 4A. When a single support labeled with a first nucleic acid sequence 401 and a second nucleic acid sequence 404 is contacted with RNA derived from a single cell, RNA409 containing a sequence complementary to the second nucleic acid sequence 404 is captured by the first nucleic acid sequence 401 on the support and forms a cDNA molecule 410 through a reverse transcription reaction system, and then the cDNA molecule 410 is melted by high-temperature denaturation of the RNA and then complementarily binds to a region of a primer sequence 407 on the second nucleic acid sequence 404 near the surface of the same support; optimally, cDNA molecules 410 can bind complementarily to more than one second nucleic acid sequence 404 on the surface of the support; further, the primer sequence 407 that binds complementary to the cDNA molecule 410 can be recognized by the associated enzyme to cleave the reversible blocking site 408 and expose the 3 ' hydroxyl group of the primer sequence 407 either by RNaseH cleavage of the ribonucleic acid base, by alkaline phosphatase treatment of the 3 ' phosphate alone, by an endonuclease capable of cleaving the AP site to form the 3 ' hydroxyl group, or by USER enzyme \ AP site excising enzyme in combination with alkaline phosphatase; followed by extension by a DNA polymerase with strand displacement activity to form a first nucleic acid molecule 411 comprising at one end the complement of the first nucleic acid sequence 401 and at the other end a cell tag sequence 406; the support with the first nucleic acid molecule 411 can be used to form a complementary strand 412 by a single primer amplification method and eluted from the support for amplification of the template, or can be used as a template for further amplification by fragmenting a second nucleic acid molecule 413 directly from the support at a conditional fragmentation site X to form a double stranded nucleic acid product 414, wherein the forward and reverse amplification primers comprise all or a portion of the nucleic acid sequences of the first universal sequence nucleic acid 402 and the second universal sequence nucleic acid 405, respectively. Further, the double-stranded nucleic acid product 414 can be analyzed for the type and abundance of single-cell RNA expression by two pooling schemes, one for unbiased analysis of the expression of all RNA molecules with polyA tail, the pooling scheme is to amplify the double-stranded nucleic acid product 414 and to form a first final library 423 by a first primer 415 comprising a first sample Index417 and a second primer 419 comprising a second sample Index421, wherein the first primer 415 comprises a first nucleic acid sequence 416 compatible with a sequencer, the first sample Index417 and a first primer hybridization region 418, and the second primer 419 comprises a second nucleic acid sequence 420 compatible with a sequencer, the second sample Index421 and a second primer hybridization region 422. Such a pooling method may be replaced by other random library construction schemes that serve the same purpose, including but not limited to, transposase-interrupted pooling or random primer extension pooling. Another library construction scheme aims to analyze the expression of a target gene in a targeted manner, and can be realized by two-step multiplex PCR, such as two-step multiplex PCR using a primer pair consisting of a first gene-specific primer 424 and a second gene-specific primer 426 and a universal primer 305: the product 425 of the first multiplex PCR and the product 427 of the second multiplex PCR are finally amplified using the product 427 of the second multiplex PCR as a template to form a second final library 428 by amplification with the first primers 415 containing the first sample Index417 and the second primers 419 containing the second sample Index 421. The library constructed by targeted multiplex PCR can be used for analysis of immune repertoires, especially for analysis of full-length T cell receptor and antibody VDJ sequences. Both the first final library 423 and the second final library 428 are further used for sequencing and information analysis.
The invention further provides application of the support co-labeled with multiple nucleic acids in construction of a single-cell transcriptome library, and the cell label can label any position of an RNA chain so as to form a cDNA molecule with a cell label and a molecular label. As shown in FIG. 5A, two types of nucleic acid labeling structures are provided on a single support, where the support is beads or hydrogel beads, and the single support has at least two nucleic acid sequences, such as a combination of a first nucleic acid sequence 501 and a second nucleic acid sequence 505, or a combination of a third nucleic acid sequence 509 and a second nucleic acid sequence 505.
The first nucleic acid sequence 501 at least comprises a capture sequence 503 for capturing a nucleic acid molecule of interest, such as oligo dT with a base sequence length of 15-40, and the efficiency of RNA capture can be controlled by adjusting the number and density of the first nucleic acid sequence 501 on the support. The first nucleic acid sequence 501 also includes a polymerase extension blocking site 504 that prevents the capture sequence 503 from extending the capture nucleic acid molecule as a primer, including but not limited to LNA/XNA/3' phosphate/inverted dT/ddNTP/C3 spacer/C6 spacer/various fluorescent dyes and quenching modifications, etc. The first nucleic acid sequence 501 also includes the first universal nucleic acid sequence 502 under specific applications, and the capture efficiency of 503 can be adjusted by adjusting the sequence and length of the first universal nucleic acid sequence 502.
The second nucleic acid sequence 505 is composed of one or more of a second universal nucleic acid sequence 506, a cell tag sequence 507, and a Primer sequence 508, wherein the second universal nucleic acid sequence 506 may include a linker nucleic acid sequence that matches a sequencer, such as a Read1 Sequencing Primer or a Read2Sequencing Primer in an illumina sequencer; cell tag sequence 507 is used to label molecules derived from all mRNAs in the same cell, with the same cell tag on each support and different cell tags on different types of supports. The cell tag sequence 507 may be a random or semi-random nucleic acid sequence, such as 12bp degenerate NNNNNNNNNNNN, or may be a combination comprising a plurality of fixed nucleic acid sequences, such as 96 kinds of 8-base sequences and 96 kinds of 8-base sequences, with or without connecting nucleic acid regions between the 8-base sequences. The primer sequence 508 can be used as a primer extension to bind to the RNA template and extend into a cDNA molecule, can bind to and extend from the RNA captured by the capture sequence 503, and the primer sequence 508 can be a random or semi-random nucleic acid sequence with a length of 5-15 bases, such as 6 random degenerate bases NNNNNN, or can be used with a gene-specific sequence to enrich for a targeted region.
The third nucleic acid sequence 509 is composed of one or more of a second universal nucleic acid sequence 506, a cell tag sequence 507, a molecular tag sequence 510, and a capture sequence 503, wherein the second universal nucleic acid sequence 506 may include a linker nucleic acid sequence that matches a sequencer, such as a Read1 Sequencing Primer or a Read2Sequencing Primer in an illumina sequencer; the cell tag sequence 507 is used for labeling molecules derived from all mRNAs in the same cell, each support has the same cell tag, different supports have different cell tags, the cell tag sequence 507 can be a random or semi-random nucleic acid sequence, such as 12bp degenerate bases NNNNNNNNNNNN, or can be a combination comprising a plurality of fixed nucleic acid sequences, such as 96 kinds of 8 base sequences and a random combination of 96 kinds of 8 base sequences and 96 kinds of 8 base sequences, and a connecting nucleic acid region can be included or excluded between the 8 base sequences; the molecular tag sequence 510 is used to label each reverse transcribed cDNA molecule, and cDNA molecules reverse transcribed from different RNAs on the same support are labeled with different molecular tags, and the molecular tag 510 can be a random or semi-random nucleic acid sequence with a length of 5-20 bases, such as 9 random degenerate bases NNNNNNNNN or NNNNNNNNNV; the capture sequence 503 is used to capture a nucleic acid molecule of interest, such as oligo dT, which is a base sequence of 15-40.
FIG. 5B is a flow chart of an experiment for constructing a single-cell transcriptome library using two types of double-nucleic acid-tagged supports. Contacting the single nucleic acid-labeled support with RNA derived from a single cell, wherein the RNA512 having a sequence complementary to the capture sequence 503 is captured by the first nucleic acid sequence 501 or the third nucleic acid sequence 509 on the support, and the RNA512 captured on the surface of the support is combined with the primer sequence 508 of the second nucleic acid sequence 505 under suitable conditions to form a cDNA molecule 514 through a reverse transcription reaction system; in particular, cDNA molecules 514 include cDNA formed using third nucleic acid 509 as a primer when a support containing third nucleic acid 509 is used; the reverse transcribed support may be further cleaved to remove the first 501, third 509 and second 505 nucleic acid sequence molecules which are not involved in the reaction. The support containing the cDNA molecules 514 can then be analyzed for the type and abundance of single cell RNA expression by two pooling approaches. One of the library construction methods, which aims to analyze the expression of all RNA molecules with polyA tail without bias, employs a random primer 517 to extend the amplification protocol. The random primer 517 consists of a universal primer sequence 515 and a random base sequence 516: the universal Primer sequences 515 may include linker nucleic acid sequences that match a sequencer, such as Read2Sequencing Primer or Read1 Sequencing Primer in an illumina sequencer; the random base sequence 516 can be a random or semi-random nucleic acid sequence of 5-15 bases in length, such as 9 contiguous degenerate bases NNNNNNNNN. Hybridizing a random primer 517 to the cDNA molecule 514 in an appropriate environment and producing a complementary strand 518 of the cDNA molecule 514 by the action of a DNA polymerase having strand displacement activity; the complementary strand 518 can be eluted from the support and amplified by a primer pair comprising the second universal nucleic acid sequence 506 and the universal primer sequence 515 to produce a double-stranded product 519; the double-stranded product 519 is amplified by a first primer 520 containing a first sample Index522 and a second primer 524 containing a second sample Index526 to form a first final library 528, wherein the first primer 520 comprises a first nucleic acid sequence 521 compatible with a sequencer, the first sample Index522 and a first primer hybridization region 523, and the second primer 524 comprises a second nucleic acid sequence 525 compatible with the sequencer, the second sample Index526 and a partial sequence of a second primer hybridization region 527; such a library construction method may be replaced with other random library construction schemes that serve the same purpose, including but not limited to, sonication, enzymatic or transposase approaches. Another library construction scheme aims to analyze the expression of a target gene in a targeted manner, and can be realized by two-step multiplex PCR, such as two-step multiplex PCR products by using a primer pair consisting of a first gene-specific primer 529, a second gene-specific primer 531 and a universal primer 506 respectively: the product 530 of the first multiplex PCR and the product 532 of the second multiplex PCR are finally amplified by the first primer 520 and the second primer 524 using the product 532 of the second multiplex PCR as a template to form a second final library 533. The library constructed by targeted multiplex PCR can be used for analysis of immune repertoires, especially for analysis of full-length T cell receptor and antibody VDJ sequences. Both the first final library 528 and the second final library 533 are further used for sequencing and information analysis.
The invention also provides the application of the support co-labeled by various nucleic acids in single cell multiomic research. According to the biological center rule, DNA carrying genetic information transfers information to RNA by transcription and is translated into protein, which finally performs major biological functions. However, since the expression amounts of RNA and protein are not consistent due to the complexity of physiological systems, and RNA does not directly reflect post-translational modification and interaction of proteins, it is important to simultaneously study the expression amounts of RNA and protein in the same cell. The present invention discloses a nucleic acid labeling support structure capable of simultaneously analyzing RNA expression level and sequence and protein expression and interaction.
As shown in fig. 6A, the support is microbeads (including solid microbeads or semi-solid hydrogel microbeads), and at least three nucleic acid sequences are labeled on the support: a first nucleic acid sequence 601, a second nucleic acid sequence 604 and a third nucleic acid sequence 609.
The first nucleic acid sequence 601 at least comprises a capture sequence 603 for capturing a nucleic acid molecule of interest and extending as a primer, such as oligo dT with a base sequence length of 15-40, the efficiency of capturing RNA can be controlled by adjusting the number and density of the first nucleic acid sequence 601 on the support; the first nucleic acid sequence 601 also includes a first universal nucleic acid sequence 602 and a conditional cleavage site X for a particular use, including but not limited to disulfide modification, dU modification, RNA base modification, dI modification, DSpacer modification, AP site modification, photocleavage PC linker, and restriction endonuclease recognition sequence.
The second nucleic acid sequence 604 is composed of one or more of a second universal nucleic acid sequence 605, a cell tag sequence 606, a molecular tag sequence 607, and a template switching sequence 608, wherein the second universal nucleic acid sequence 605 may include a linker nucleic acid sequence that matches a sequencer, such as a Read1 Sequencing Primer or a Read2Sequencing Primer in an illumina sequencer; the cell tag sequence 606 is used to label all mRNA molecules from the same cell, each support has the same cell tag and different supports have different cell tags, the cell tag sequence 606 can be a random or semi-random nucleic acid sequence, such as 12bp degenerate bases NNNNNNNNNNNN, or a combination comprising multiple fixed nucleic acid sequences, such as 96 8 base sequences and a random combination of 96 8 base sequences and 96 8 base sequences, and the 8 base sequences may or may not include a linking nucleic acid region; the molecular tag sequence 607 is used to label each reverse transcribed cDNA molecule, and cDNA molecules reverse transcribed from different RNAs on the same support are labeled with different molecular tags, and the molecular tag 607 can be a random or semi-random nucleic acid sequence with a length of 8-20 bases, such as 9 random degenerate bases NNNNNNNNN or NNNNNNNNNV; the template switch sequence 608 can serve as a template to extend the 3 'end of the cDNA reverse transcribed from the first nucleic acid sequence 601 to label the molecular tag sequence 607, the cellular tag sequence 606, and the second universal nucleic acid sequence 605, and the template switch sequence 608 includes two or more RNA bases rG or other modified base G analogs, such as LNA or XNA, at least at the 3' end.
The third nucleic acid sequence 609 consists of one or more of a third universal nucleic acid sequence 610, a cell tag sequence 606, a molecular tag sequence 607 and a protein nucleic acid tag capture sequence 611, wherein the cell tag sequence 606 and the molecular tag sequence 607 are identical to the structure on the second nucleic acid sequence 604; the third universal nucleic acid sequence 610 is a nucleic acid sequence that does not correspond to the second universal nucleic acid sequence 605 and contains a linker that matches a sequencer, such as Read1 Sequencing Primer or Read2Sequencing Primer in an illumina sequencer; the protein nucleic acid tag capture sequence 611 is used to capture and extend a protein nucleic acid tag in the same spatial structure with the single cell to be detected, and the same spatial structure means that the protein nucleic acid tag can be located inside the cell, on the surface of the cell, or in a chamber or a droplet where the cell is located.
FIG. 6B is a flow chart of an experiment for constructing a multi-genomic single-cell library from the three nucleic acid-labeled supports shown in FIG. 6A. Firstly, a cell to be detected is contacted and combined with an antibody molecule 612 which is coupled with a nucleic acid label in advance and can identify specific protein, and the nucleic acid coupled antibody which is not specifically combined is washed away, wherein the structure of the antibody molecule 612 comprises a sequence 613 which can be complementarily combined with a protein nucleic acid label capture sequence 611, a protein specific sequence 614, a fourth Primer sequence 615 and a molecule 616, the fourth Primer sequence 615 is a linker nucleic acid sequence which is inconsistent with the second general nucleic acid sequence 605 and the third general nucleic acid sequence 610 and contains a linker nucleic acid sequence matched with a sequencer, such as a Read2Sequencing Primer or a Read1 Sequencing Primer in an illumina sequencer, and the molecule 616 refers to a specific antibody in the flow and can also be other substances such as small molecular compounds, saccharides, peptides and the like which are mutually combined with target detection protein; when a single support is contacted with single cells bound with nucleic acid-coupled antibody molecules 612, the cells are lysed to release RNA617 and nucleic acid-coupled antibody molecules 612 and captured by the first nucleic acid sequence 601 and the third nucleic acid sequence 609 on the support, respectively, and cDNA molecules 618 or DNA molecules 619 are formed by a reverse transcription reaction system, wherein the cDNA extends to the 5' end of the RNA617, and is added with continuous bases C to a cDNA chain by a reverse transcriptase having a terminal nucleotidyl transferase function, and then the cDNA chain is complementarily bound with a template switching sequence 608 containing more than two bases rG or base analogues thereof near the surface of the same support and extends to the second universal nucleic acid sequence 605 area to form complete cDNA molecules 618 with cell tags and molecular tags; alternatively, cDNA molecules 618 and DNA molecules 619 may be detached from the support at cleavable sites X as a template for the next amplification step, or an extended strand complementary to cDNA molecules 618 or DNA molecules 619 formed by single primer extension of second universal nucleic acid sequence 605 and fourth universal nucleic acid sequence 615 may be used as a template for the next amplification step, or a support containing cDNA molecules 618 and 619 may be used as a template for the next amplification step after enzymatic treatment to remove first nucleic acid sequence 601, second nucleic acid sequence 604 and third nucleic acid sequence 609 that are not involved in the reverse transcription reaction from the support; in a subsequent step, the mixture of fragmented or non-fragmented cDNA molecules 618 and DNA molecules 619 is PCR amplified as a template with a first universal nucleic acid sequence 602/second universal nucleic acid sequence 605 and a third universal nucleic acid sequence 610/fourth universal nucleic acid sequence 615 double primer pair to form a mixture of a first double stranded nucleic acid product 621 and a second double stranded nucleic acid product 620. Further, the formed double-stranded nucleic acid product can be subjected to single-cell multiomic analysis through three library establishing modes. The first library construction scheme aims to construct a nucleic acid library capable of analyzing the abundance of a protein to be detected, and a first library 630 can be obtained by directly performing PCR amplification on a first index primer 622 and a second index primer 626; wherein the first index primer 622 comprises a first sequencer-compatible nucleic acid sequence 623, a first sample index624, and a nucleic acid sequence 625 complementary to the fourth universal nucleic acid sequence 615, which are linked in sequence, and the second index primer 626 comprises a second sequencer-compatible nucleic acid sequence 627, a second sample index628, and a nucleic acid sequence 629 complementary to the third universal nucleic acid sequence 610, which are linked in sequence. The objective of the second pooling scheme, which is to randomly break the mixture of the first double-stranded nucleic acid product and the second double-stranded nucleic acid product, repair the ends and add base A at the 3' end to form a molecular structure 631, then connect with the linker 632 containing the protruding T and amplify by the first primer 634 containing the first sample Index624 and the second primer 636 containing the second sample Index628 to form the second final library 638, wherein the first primer comprises the first nucleic acid sequence 623 compatible with the sequencer, the first sample Index624 and the nucleic acid sequence 635 complementary to the first universal nucleic acid sequence 602 connected in sequence, the second primer 636 comprises the second nucleic acid sequence 627 compatible with the sequencer, the second sample Index628 and the nucleic acid sequence 637 complementary to the second universal nucleic acid sequence 605 connected in sequence, is to analyze the expression of all RNA molecules with polyA tail without bias, and this pooling method can be replaced by other random pooling schemes capable of achieving the same objective, including but not limited to, a transposase method disruption pooling or a random primer extension pooling scheme. The purpose of the third library construction scheme is to perform targeted analysis on the expression of the target gene, which can be achieved by two-step multiplex PCR, such as forming a multiplex PCR product by using a primer pair consisting of a first gene-specific primer 639 and a second gene-specific primer 641, and a second universal primer 605: first multiplex PCR product 640 and second multiplex PCR product 642, and finally second multiplex PCR product 642 as a template, are amplified with first primers 634 containing first sample Index624 and second primers 636 containing second sample Index628 to form third final library 643. The library constructed by targeted multiplex PCR can be used for analysis of immune repertoires, and further, the second multiplex PCR product 642 can be used for constructing a full-length VDJ immune repertoire library for analysis of T cell receptor and antibody VDJ sequences according to the first random break library construction scheme. Library 630, second final library 638, and third final library 643 are all further used for sequencing and information analysis.
Example 1 application of support co-labeled with multiple nucleic acids to 5' Single-cell RNA expression profiling library construction and VDJ library construction and Multi-group chemical library construction
In this example, various nucleic acid co-labeled supports were prepared and used to construct 5' single cell RNA expression profiling libraries, VDJ libraries, and multigenomic libraries according to the following procedures.
1 preparation of magnetic beads labeled with various nucleic acids
1.1 Synthesis of the following sequence of single-stranded nucleic acids.
Figure BDA0002996416330000251
1.2 at 0.25M EDC concentration, 384 kinds of 300pmol amino-modified CB1 single-stranded nucleic acid (SEQ ID No.2), 300pmol amino-modified dT single-stranded nucleic acid (SEQ ID No.1) and 6 ten thousand 30. mu.M magnetic beads were mixed at room temperature by rotation for 3 hours, and after washing twice, 384 kinds of nucleic acid-labeled magnetic beads were obtained.
1.3 the 384 nucleic acid labeled magnetic beads obtained in 1.2 were mixed uniformly and then divided into 384 well plates.
1.4 annealing of single stranded nucleic acid CB2-TSO (SEQ ID No.3, 3 'end of which 3 "(rG)" is G of RNA) with the same Cell Barcode together with CB2-T7(SEQ ID No.5) and rCB (SEQ ID No.4) to form a double stranded structure with sticky ends, where the sequence n' n 'n' n 'n' CTGTAG in rCB2 is the reverse complement of CTACAGnnnnnnnnnnnn in CB2-TSO and CB 2-T7; nnnnnnnnnn is an 8bp Cell barcode sequence, and in this example, there are a total of 384 types.
1.5 Add 384 annealed double-stranded nucleic acids CB2-rCB2 to 384-well plate containing magnetic beads in the following ratio, and react at 22 ℃ for 30 minutes.
Reagent 50 μ L system
2X Rapid Ligation Buffer 25μL
CB2-rCB2 double-stranded nucleic acid 3μL
T4 DNA Ligase 3μL
RNase-free water Make up to 50 μ L
1.6 after the reaction, 384 magnetic beads are evenly mixed, and the mixture is treated at a high temperature of 95 ℃ to remove complementary strands and then placed at 4 ℃ for standby.
2 labeling single-cell cDNA and protein by using multi-nucleic acid labeling magnetic beads
2.1 antibodies against human CD4 molecule coupled to T7 nucleic acid sequence were mixed with fresh PBMC cells and incubated to allow for sufficient binding of the antibodies to cell membrane surface CD4 protein, followed by washing with fresh PBS.
2.2 the chips were processed according to the microwell chip instructions provided in the BD Rhapbody Single cell sequencing kit and 1 ten thousand incubated PBMC cells were added.
2.3 adding 30 ten thousand magnetic beads prepared in the step 1 into the microporous plate, and cleaning redundant magnetic beads after magnetically attracting the holes.
2.4 adding the lysis solution in the kit, and after 2min, taking out the magnetic beads by magnetic attraction and cleaning.
2.5A reverse transcription reagent (200 uL) was prepared and magnetic beads were suspended in the following reaction.
Reagent 200 μ L system
Superscript II first-strand buffer(5×) 40
DTT(100mM) 10
Betaine(5M) 40
MgCl2(1M) 1.2
dNTP 10mM 20
RNAse inhibitor 5
SuperScript II reverse transcriptase 10
RNase-free water 73.8
2.6 reverse transcription was performed under the following conditions.
Figure BDA0002996416330000261
2.7 directly removing the reverse transcription supernatant and adding the following 200uL exonuclease reaction solution, and reacting at 37 ℃ for 30 minutes to remove the redundant primers on the magnetic beads.
Reagent 200 μ L system
10 Xexonuclease buffer 20μL
Exo-enzyme 10μL
RNase-free water 170μL
3 constructing high-throughput sequencing library of single cell membrane protein expression quantity
3.1 washing the magnetic beads by using an elution buffer solution carried by a BD Rhapbody kit, resuspending, treating at the high temperature of 95 ℃ for 5min, and immediately sucking out a supernatant for later use; the remaining beads were resuspended in elution buffer for use.
3.2 configure the following PCR Mix.
Components For 1library(μL)
PCR MasterMix(Cat.No.91-1118) 100
Universal Oligo(Cat.No.650000074) 10
Bead RT/PCR Enhancer(Cat.No.91-1082) 12
Primer T7(SEQ ID No.7) 10
2.2.8 elution of supernatant 68
Total 200
3.3 amplification was performed using SPRI beads 1.4 Xpurification and 30uL elution under the following conditions.
Figure BDA0002996416330000271
3.4 the following Index PCR mix was prepared.
PCR MasterMix(Cat.No.91-1118) 25
Index P5 primer(SEQ ID No.9) 2
Index P7 primer(SEQ ID No.10) 2
Nuclease-free water 18
2.3.3 eluent in 3
Total 50
3.5 after amplification according to the following conditions, SPRI beads 0.8 x are used for purification, 30uL sterile water is used for elution to obtain a single cell expression library of the membrane protein CD4, the size of the library fragment is about 280bp, and the library detection standard is met, as shown in FIG. 7A.
Figure BDA0002996416330000272
4 single cell 5' expression profile library and construction of immune receptor VDJ library
4.1 full-Length cDNA amplification: the following PCR reaction was configured and the magnetic beads prepared in 2.3.1 were resuspended.
Kit Components For 1library(μL)
PCR MasterMix(Cat.No.91-1118) 60
Universal Oligo(Cat.No.650000074) 10
Primer Full(SEQ ID No.8) 10
RNase-free water 40
Total 120
4.2 after amplification using SPRI beads 0.6 Xpurification, 30uL elution was performed for future use.
Figure BDA0002996416330000281
4.3 the partial eluate in 4.2 was pooled by using the 5 'Library Construction Kit (PN-1000020) of the chromosome Single Cell V (D) J Reagent Kit of 10 Xgenomics to obtain a 5' Single Cell expression profile Library, as shown in FIG. 7B, which shows that the main peak of the constructed Library is in the vicinity of 484, which meets the Library check standard.
4.4 the fraction eluted in 4.2 was amplified and pooled using the Enrichment Kit (Human T Cell, PN-1000005 and Human B Cell, PN-1000016) from the chromosome Single Cell V (D) J Reagent Kit of 10 × genomics to obtain Single Cell VDJ libraries of T cells and B cells, as shown in FIGS. 7C and 7D, respectively. As can be seen in FIG. 7C, the size of the constructed library was between 200 and 1000bp, which is expected. As can be seen in FIG. 7D, the main peak of the constructed library is near 545, and meets the library test standard.
Example 2: application of support co-labeled with multiple nucleic acids to 3' single-cell RNA library construction
In this example, various nucleic acid co-labeled supports were prepared and used to construct 3' single cell RNA libraries according to the following protocol.
1 preparation of magnetic beads labeled with various nucleic acids
1.1 Synthesis of the following sequence of single-stranded nucleic acids.
Figure BDA0002996416330000282
1.2 at 0.25M EDC concentration, 384 kinds of 300pmol amino-modified CB1 single-stranded nucleic acid (SEQ ID No.2), 300pmol amino-modified SP2-dT30VN single-stranded nucleic acid (SEQ ID No.11) and 6 ten thousand 30. mu.M magnetic beads were mixed at room temperature by rotation for 3 hours, and the mixture was washed twice to obtain 384 kinds of nucleic acid-labeled magnetic beads.
1.3 the 384 nucleic acid labeled magnetic beads obtained in 1.2 were mixed uniformly and then divided into 384 well plates.
1.4 annealing of a single stranded nucleic acid CB2-NrNx (SEQ ID No.12, wherein "(rN)" represents an RNA base and further carrying a C3 modification at the 3 'end of the sequence) to rCB2 forms a double stranded structure with sticky ends, wherein the sequence n' n 'n' n 'n' n 'n' CTGTAG in rCB2 is the reverse complement of CTACAGnnnnnnnnnnnn in CB 2-NrNx; nnnnnnnnnn is an 8bp Cell barcode sequence, sharing 384 types.
1.5 Add 384 annealed CB2-NrNx/rCB2 double-stranded nucleic acids to a 384-well plate containing magnetic beads in the following ratio, and react at 22 ℃ for 30 minutes.
Reagent 50 μ L system
2X Rapid Ligation Buffer 25μL
CB2-NrNx/rCB2 double-stranded nucleic acid 3μL
T4 DNA Ligase 3μL
RNase-free water Make up to 50 μ L
1.6 after the reaction, 384 magnetic beads are evenly mixed, and the mixture is treated at a high temperature of 95 ℃ to remove complementary strands and then placed at 4 ℃ for standby.
2 3' Single-cell RNA library construction Using Multi-nucleic acid-labeled magnetic beads
2.1 peripheral blood was drawn and fresh PBMC cells were obtained and resuspended in PBS.
2.2 the chips were processed according to the microwell chip instructions provided in the BD Rhapbody Single cell library construction kit and 1 ten thousand incubated PBMC cells were added.
2.3 adding 30 ten thousand magnetic beads prepared in the step 2.1 into the microporous plate, and cleaning redundant magnetic beads after magnetically attracting the holes.
2.4 adding the lysis solution in the kit, and after 2min, taking out the magnetic beads by magnetic attraction and cleaning.
2.5 reverse transcription and ExoI cleavage of unused primers on magnetic beads as described in the BD Rhapbody Single cell library construction kit.
2.6 random primer NrNx duplex extension: the following hybridization reaction system was prepared and the magnetic beads obtained in 3.2.5 were suspended.
Kit Components For 1 library(μL)
WTA Extension Buffer(Cat.No.91-1114) 20
Nuclease-free water(Cat.No.650000076) 150
RNase HII 4
Total 174
2.7 hybridization was carried out under the following temperature conditions.
Figure BDA0002996416330000291
2.8 the following extension reagents were added.
Kit Components For 1 library(μL)
10mM dNTP(Cat.No.650000077) 8
Bead RT/PCR Enhancer(Cat.No.91-1082) 12
WTA Extension Enzyme(Cat.No.91-1117) 6
Total 26
2.9 extension according to BD kit conditions (conditions below).
Figure BDA0002996416330000301
2.10 after the extension, the magnetic beads were washed, and the following reaction system was prepared and suspended.
Kit Components For 1library(μL)
PCR MasterMix(Cat.No.91-1118) 60
Universal Oligo(Cat.No.650000074) 10
UPP-2(SEQ ID No.13) 10
RNase-free water 40
2.11 PCR was performed according to the following reaction conditions, followed by purification using SPRI beads 0.9X and elution at 30 uL.
Figure BDA0002996416330000302
2.12 the following Index PCR mix was configured.
PCR MasterMix(Cat.No.91-1118) 25
Index P5 primer 2
Index P7 primer 2
Nuclease-free water 11
3.2.11 from the eluent 10
Total 50
2.13 amplification Using SPRI beads 0.5/0.25 Xsorting, elution with 30uL of sterile water gave a 3' single-cell RNA library.
Figure BDA0002996416330000303
2.14 illumina NovaSeq 6000 and analyzing the location of the detected nucleic acid sequence in the RNA, the detected nucleic acid sequence is located predominantly near the 3' end of the RNA as shown in FIG. 8.
Example 3: application of support co-labeled with multiple nucleic acids in construction of single-cell transcriptome library
In this example, various nucleic acid co-labeled supports were prepared and used to construct a single-cell transcriptome library according to the following procedures.
1 preparation of magnetic beads labeled with various nucleic acids
1.1 Synthesis of the following sequence of single-stranded nucleic acids.
SEQ ID No. Name (R) Sequence of
14 CB2-dN6 CTACAGnnnnnnnnNNNNNN
1.2 at 0.25M EDC concentration, 384 kinds of 300pmol amino-modified CB1 single-stranded nucleic acid (SEQ ID No.2), 300pmol amino-modified dT single-stranded nucleic acid (SEQ ID No.1) and 6 ten thousand 30. mu.M magnetic beads were mixed at room temperature by rotation for 3 hours, and after washing twice, 384 kinds of nucleic acid-labeled magnetic beads were obtained.
1.3 the 384 nucleic acid labeled magnetic beads obtained in 1.2 were mixed uniformly and then divided into 384 well plates.
1.4 annealing of single stranded nucleic acid CB2-dN6(SEQ ID No.14) to rCB2 forms a double stranded structure with sticky ends, wherein the sequence n 'n' n 'n' n 'n' CTGTAG in rCB2 is the reverse complement of CTACAGnnnnnnnn in CB2-dN 6; nnnnnnnnnn is an 8bp Cell barcode sequence, sharing 384 types.
1.5 the 384 annealed double-stranded nucleic acids CB2-dN6/rCB2 were added to a 384-well plate containing magnetic beads in the following ratio, and reacted at 22 ℃ for 30 minutes, respectively.
Reagent 50 μ L system
2X Rapid Ligation Buffer 25μL
CB2-dN6/rCB2 double-stranded nucleic acid 3μL
T4 DNA Ligase 3μL
RNase-free water Make up to 50 μ L
1.6 after the reaction, 384 magnetic beads are evenly mixed, and the mixture is treated at a high temperature of 95 ℃ to remove complementary strands and then placed at 4 ℃ for standby.
2 Single cell transcriptome library construction Using Multi-nucleic acid-labeled magnetic beads
2.1 peripheral blood was drawn and fresh PBMC cells were obtained and resuspended in PBS.
2.2 the chips were processed according to the microwell chip instructions provided in the BD Rhapbody Single cell library construction kit and 1 ten thousand incubated PBMC cells were added.
2.3 adding 30 ten thousand magnetic beads prepared in the step 1 into the microporous plate, and cleaning redundant magnetic beads after magnetically attracting the holes.
2.4 adding the lysis solution in the kit, and after 2min, taking out the magnetic beads by magnetic attraction and cleaning.
2.5 reverse transcription and ExoI excision of unused primers on magnetic beads as described in the BD Rhapbody Single cell library construction kit, with reverse transcription reaction conditions modified as follows:
Figure BDA0002996416330000311
Figure BDA0002996416330000321
2.6 the following steps were followed to generate the final Library by hybridization extension and amplification exactly as described in BD Rhapbody mRNA White Transcription Analysis (WTA) Library Preparation Protocol.
2.7illumina NovaSeq 6000, and analyzing the position of the detected nucleic acid sequence in the RNA.
The nucleic acid sequences determined by this method are more evenly distributed over the entire length of the RNA than the BD rhapsody and the library of example 2. As shown in FIG. 8, the distribution of reads at the gene level was obtained by sequencing analysis using the 3' single-cell RNA library constructed by the procedure in example 2 and the single-cell transcriptome library constructed by the procedure in example 3. The BD Phapsody 3' single cell expression profiling library was a library analysis structure constructed completely using BD Rhapsody. As can be seen from the figure, the sequences contained in the 3 'single cell RNA library constructed in example 2 were mainly distributed at the 3' end of the gene, whereas the sequences contained in the single cell transcriptome library constructed in example 3 were significantly more biased toward the middle position of the gene than the 3 'single cell RNA library and the BD Phapsody 3' single cell expression profile library.
Example 4: application of support co-labeled with multiple nucleic acids in construction of multiple PCR sequencing libraries
The purpose of this example is to realize single-tube multiplex PCR detection of full-length gene sequences of Brca1 and Brca2, and the designed multiplex PCR primers are as follows:
Pool 1：SEQ ID No.15～SEQ ID No.34；
Pool 2：SEQ ID No.35～SEQ ID No.54；
Pool 3：SEQ ID No.55～SEQ ID No.74；
Pool 4：SEQ ID No.75～SEQ ID No.94；
Pool 5：SEQ ID No.95～SEQ ID No.114；
Pool 6：SEQ ID No.115～SEQ ID No.134；
Pool 7：SEQ ID No.135～SEQ ID No.154；
Pool 8：SEQ ID No.155～SEQ ID No.174；
Pool 9：SEQ ID No.175～SEQ ID No.194；
Pool 10：SEQ ID No.195～SEQ ID No.214；
Pool 11：SEQ ID No.215～SEQ ID No.234；
Pool 12：SEQ ID No.235～SEQ ID No.254；
Pool 13：SEQ ID No.255～SEQ ID No.274；
Pool 14：SEQ ID No.275～SEQ ID No.294；
Pool 15：SEQ ID No.295～SEQ ID No.314；
Pool 16：SEQ ID No.315～SEQ ID No.334；
Pool 17：SEQ ID No.335～SEQ ID No.348。
1 extraction of genomic DNA
200uL of human peripheral blood genomic DNA was extracted according to the Tiangen blood genomic DNA extraction kit instructions and the concentration was measured using a Qubit.
2 preparation of magnetic beads labeled with Polynucleic acids
2.1 Synthesis of 5' amino modified primer sequences according to the sequences in Table 1 and mixing together equal amounts of primers with the same pool number.
2.2 mix 10nmol of primer mixture of Pool numbers 1-17 (Pool 1, Pool 2, Pool 3, Pool 4, Pool 5, Pool 6, Pool 7, Pool 8, Pool 9, Pool 10, Pool 11, Pool 12, Pool 13, Pool 14, Pool 15, Pool 16 or Pool 17) with 10mg of magnetic beads (Dynabeads MyOne Carboxylic Acid) at room temperature for 3 hours under 0.25M EDC concentration, and wash twice to obtain 17 kinds of nucleic Acid labeled magnetic beads.
2.3 mixing the obtained 17 kinds of nucleic acid labeled magnetic beads according to the proportion of 1:1 for later use.
3 multiplex PCR amplification
3.1 PCR system was configured as per the following table.
Reagent 50 μ L system
2x QIAGEN Multiplex PCR Master Mix 25μL
Nucleic acid-labeled magnetic beads 5μL
Genomic DNA 10ng
RNase-free water Make up to 50 μ L
3.2 run on a PCR machine according to the following procedure.
Figure BDA0002996416330000331
4Index PCR-generated libraries
4.1 index PCR system was prepared according to the following table.
Reagent 50 μ L system
2x KAPA Hifi 25μL
I5 Primer 5μL
I7 Primer 5μL
RNase-free water Make up to 50 μ L
4.2 suspend washed magnetic beads in 1.3.2 with a prepared 50. mu.L index PCR system, run the following PCR program:
Figure BDA0002996416330000341
4.3 use SPRI beads to purify DNA with length of 300-500bp, measure concentration and use Caliper to analyze fragment length, as shown in FIG. 9, obtain library with main peak of about 379bp, which meets the library detection standard.

Claims (15)

1. A multi-nucleic acid co-labeled support comprising a support body and a plurality of nucleic acid labels located on the surface and/or within the support body, wherein the labeled nucleic acids on a single support comprise at least: one or more first nucleic acid labels, the function of which at least comprises capturing a specific compound in the reaction system to the surface of the support; one or more second nucleic acid labels, which function at least comprises a defined biochemical reaction process that can take part in a specific compound captured to the surface of the support.
2. The plurality of nucleic acid co-labeled supports of claim 1, wherein the support bodies are solid beads and/or semi-solid hydrogel beads.
3. The plurality of nucleic acid co-labeled supports of claim 1, which is a composition comprising a plurality of supports.
4. The support co-labeled with a plurality of nucleic acids according to claim 3, wherein the number of the first nucleic acid label and the number of the second nucleic acid label on the same support are 1 or more and/or 10 or less, respectively13And (4) respectively.
5. The multiple nucleic acid co-labeled support according to claim 3, wherein,
the sequences of the plurality of first nucleic acid tags on the same support may be the same or different;
the sequences of the first nucleic acid tags on different supports are the same or different;
the sequences of the plurality of second nucleic acid tags on the same support may be the same or different; or
The sequences of the second nucleic acid tags on different supports may be the same or different.
6. A method for preparing a support co-labeled with a plurality of nucleic acids according to any one of claims 1 to 5, comprising:
a plurality of nucleic acids are labeled onto the support body by grafting and/or grafting to obtain a plurality of nucleic acid co-labeled supports.
7. The method of fabrication of claim 6, comprising:
modifying the support body and the nucleic acid with functional units capable of interacting with each other, respectively, so that the two react to label the nucleic acid on the support body;
synthesizing nucleic acid directly on a support body according to a preset nucleotide sequence; and/or
Nucleic acid labeling is carried out on the support body using a protocol of biochemical reactions for nucleic acid extension or attachment.
8. Use of the plurality of nucleic acid co-labeled supports of any one of claims 1-5 for 5 ' single cell RNA expression profiling, constructing a5 ' single cell VDJ library for a microwell array platform, constructing a3 ' single cell RNA library, constructing a single cell transcriptome library, single cell multigroup studies, multiplex PCR, and/or constructing a multiplex PCR sequencing library.
9. The use of claim 8, wherein said plurality of nucleic acid co-labeled supports are used for 5' single cell RNA expression profiling: wherein:
the support is fixed with a template switching sequence containing a cell label and a molecular label and an RNA capture sequence, and specifically, the support is marked with at least two nucleic acid sequences: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending or reverse transcribing as a primer; the second nucleic acid sequence comprises a cell tag sequence for tagging molecules derived from all mRNAs in the same cell; different cell labels on different kinds of supports;
preferably, the support is allowed to capture RNA released after single cell lysis in the microwells of the chip, and labeling of RNA derived from the same cell with the same cell tag is achieved by template conversion during reverse transcription, followed by amplification of cDNA and final construction into a library of 5' single cell RNA expression profiles.
10. The use of claim 8, wherein said plurality of nucleic acid co-labeled supports is a 5' single cell VDJ library for constructing a microwell array platform; wherein:
the support is fixed with a template switching sequence containing a cell label and a molecular label and an RNA capture sequence, and specifically, the support is marked with at least two nucleic acid sequences: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending or reverse transcribing as a primer; the second nucleic acid sequence comprises a cell tag sequence, a molecular tag sequence and a template switching sequence, wherein the cell tag sequence is used for marking molecules derived from all mRNA in the same cell; the molecular tag sequence is used for marking each reverse-transcribed cDNA molecule, and cDNA molecules reverse-transcribed from different RNAs on the same support are marked with different molecular tags; the template conversion sequence can be used as a template to continue extending the 3' end of the reverse transcribed cDNA so as to mark a molecular tag sequence and a cell tag sequence; different cell labels on different kinds of supports;
preferably, the support captures RNA released after single cell lysis in the micropores of the chip, and labeling RNA derived from the same cell with the same cell label is realized through template conversion in the reverse transcription process, and enrichment of TCR and BCR/Ig nucleic acid sequences is further realized through TCR and BCR/Ig gene constant region primers, and finally the construction of a high-throughput single-cell VDJ sequencing library is interrupted.
11. The use of claim 8, wherein said plurality of nucleic acid co-labeled supports are used to construct a 3' single cell RNA library; wherein:
the support has immobilized thereon conditionally-encapsivable random primers comprising a cell tag and an RNA capture sequence, and in particular, the support is labeled with at least two nucleic acid sequences: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending or reverse transcribing as a primer; the second nucleic acid sequence comprises a conditionally-closed random primer comprising a cell tag sequence for labeling molecules derived from all mRNAs in the same cell; different cell labels on different kinds of supports;
preferably, the support is allowed to capture RNA released after single cell lysis in the microwells of the chip and reverse transcribe into cDNA, subsequent random primers containing cell tags effect the same cell tag on cDNA derived from the same cell by two-strand synthesis, and cDNA amplification is then achieved by amplification to construct a 3' single cell RNA library.
12. The use of claim 8, wherein said plurality of nucleic acid co-labeled supports are used to construct a single cell transcriptome library; wherein:
random primer sequences containing cell labels and RNA capture sequences are fixed on the supports, different cell labels are arranged on different kinds of supports, and any sequence on the RNA molecule can be detected without being limited to the 3 'end or the 5' end; preferably, the support comprises two types of supports, each type of support having at least two nucleic acid sequences, a combination of a first nucleic acid sequence and a second nucleic acid sequence, or a combination of a third nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence comprises at least a capture sequence for capturing a nucleic acid molecule of interest; the second nucleic acid sequence comprises a random primer sequence comprising a cell tag sequence for labeling molecules derived from all mRNAs in the same cell; the third nucleic acid sequence comprises a cell tag sequence and a capture sequence;
preferably, the support is made to capture the RNA released after single cell lysis in the microwells of the chip and to label the same cell tags for RNA originating from the same cells during reverse transcription, followed by amplification of cDNA and final construction into a single cell RNA transcriptome library.
13. The use of claim 8, wherein said plurality of nucleic acid co-labeled supports are used for single cell multigroup studies; preferably, nucleic acid tags for constructing a library of RNA expression levels and/or for detecting protein expression levels by proteins are included; wherein:
the support is fixed with an RNA capture sequence containing a cell label and a capture sequence of a nucleic acid label for marking protein, and different types of supports have different cell labels; preferably, the first nucleic acid sequence comprises at least a capture sequence for capturing the nucleic acid molecule of interest and extending as a primer; the second nucleic acid sequence comprises a cell tag sequence, a molecular tag sequence and a template switching sequence; cell tag sequences are used to label molecules derived from all mrnas in the same cell; the molecular tag sequence is used for marking each reverse-transcribed cDNA molecule, and cDNA molecules reverse-transcribed from different RNAs on the same support are marked with different molecular tags; the template conversion sequence can be used as a template to continue extending the 3' end of the reverse transcribed cDNA so as to mark a molecular tag sequence and a cell tag sequence; the third nucleic acid sequence comprises a cell tag sequence, a molecular tag sequence and a protein nucleic acid tag capture sequence, wherein the protein nucleic acid tag capture sequence is used for capturing and extending a protein nucleic acid marker which has the same spatial structure with the single cell to be detected;
preferably, the support captures the nucleic acid tags of RNA and protein released after single cell lysis in the micropores of the chip, and the same cell tags are marked on the RNA and protein nucleic acid tags from the same cell in the reverse transcription process, and then the RNA and protein nucleic acid tags are finally constructed into a single cell RNA transcriptome library and a protein marker nucleic acid library through amplification.
14. The use of claim 8, wherein said plurality of nucleic acid co-labeled supports are used to construct a multiplex PCR sequencing library; wherein:
immobilizing primers capable of interfering with each other to different supports, respectively; specifically, the support comprises at least two types of supports: one or more first species of primer-labeled supports, one or more second species of primer-labeled supports, each support having at least one pair of nucleic acid primers labeled thereon: the first kind of primer-labeled support is labeled with a first nucleic acid primer pair, the second kind of primer-labeled support is labeled with a second nucleic acid primer pair different from the first nucleic acid primer pair, each of the two kinds of supports independently optionally comprises more nucleic acid primer pairs such as other nucleic acid primer pairs, and target fragments amplified by the primer pairs on the same support do not coincide on the template; the primer pairs labeled on different supports are different so as to amplify different target regions, and the target regions can be partially overlapped or not overlapped;
preferably, all the supports are mixed in proportion and then mixed with the nucleic acid template and the PCR enzyme reaction system, thereby performing single-tube unbiased multiplex PCR.
15. A kit comprising a plurality of nucleic acid co-labeled supports according to any one of claims 1 to 5;
preferably, the kit is a kit applicable to 5 ' single cell RNA expression profiling, 5 ' single cell VDJ library construction of a microwell array platform, 3 ' single cell RNA library construction, single cell transcriptome library construction, single cell multigroup study, multiplex PCR and/or multiplex PCR sequencing library construction;
more preferably, the kit further comprises one or more of the following compositions:
composition 1: a support mixture containing a template conversion sequence of a cell label and a molecular label and an RNA capture sequence, a microporous chip, a cell lysate, a reverse transcription reagent, a nucleic acid amplification reagent and a nucleic acid interruption library building module;
composition 2: a support mixture containing a template conversion sequence of a cell label and a molecular label and an RNA capture sequence, a microporous chip, a cell lysate, a reverse transcription reagent, a constant region primer, a nucleic acid amplification reagent and a nucleic acid breaking library building module;
composition 3: a mixture of random primers containing cell tags and a support for RNA capture sequences, a microwell chip, a cell lysate, a reverse transcription reagent, a double-stranded synthesis module, and a nucleic acid amplification and extension reagent;
composition 4: a support mixture containing a random primer sequence of a cell label and an RNA capture sequence, a microporous chip, a cell lysate, a reverse transcription reagent, a double-strand synthesis module and a nucleic acid amplification and extension reagent;
composition 5: a capture sequence support mixture containing protein tag nucleic acid of a cell tag, a microporous chip, cell lysate, a reverse transcription reagent and a nucleic acid breaking library building module;
composition 6: pre-mixing the primer-coupled support mixture with multiplex PCR enzymes and buffers; further optionally, the kit further comprises a tag primer adapted to the high-throughput sequencer.
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