WO2022021279A1

WO2022021279A1 - Multi-nucleic acid co-labeling support, preparation method therefor, and application thereof

Info

Publication number: WO2022021279A1
Application number: PCT/CN2020/106089
Authority: WO
Inventors: 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
Original assignee: 北京寻因生物科技有限公司
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2022-02-03
Also published as: CN114096678A

Abstract

Provided are a variety of nucleic acid co-labeling supports, a preparation method therefor, and an application thereof. The supports comprise a support body, and a variety of nucleic acid labels located on the surface and/or inside of the support body. The nucleic acid labeled on a single support at least comprises: one or more first nucleic acid labels having a function that at least comprises of trapping a specific compound in a reaction system onto the surface of the support; and one or more second nucleic acid labels having a function that at least comprises participating in a specified biochemical reaction process of the specific compound trapped onto the surface of the support. The foregoing variety of nucleic acid co-labeled supports can be used for 5'-terminus single-cell RNA expression profile analysis, construction of a 5'-terminus single-cell VDJ library for a microwell array platform, construction of a 3'-terminus single-cell RNA library, construction of a single-cell transcriptome library, single-cell multi-omics research, multiplex PCR and/or construction of a multiplex PCR sequencing library, etc.

Description

A variety of nucleic acid co-labeling supports and their production methods and applications

technical field

The present invention relates to a kind of multiple nucleic acid co-labeling support and its preparation method and application.

Background technique

Traditional nucleic acid molecule reactions, including nucleic acid hybridization, extension, amplification and other reactions are carried out in liquid phase. The liquid phase provides a uniform and stable environment for the nucleic acid and enzyme reactions involved in the reaction to maximize the output. With the gradual deepening of biological research, scientists have found that attaching nucleic acids or enzymes involved in the reaction to the surface of the solid phase can give the nucleic acid spatial position information to facilitate purification, separation, detection and analysis. Therefore, more and more solid phase nucleic acids have been developed. The reaction is used for nucleic acid sequence analysis and nucleic acid quantification, such as nucleic acid chip technology in which oligonucleotides are immobilized on a substrate in an orderly manner, bridge amplification and microsphere emulsion amplification technology for next-generation gene sequencing, and high-throughput applications Nucleic acid encoding microbeads for single-cell sequencing, etc.

Single nucleotide polymorphisms (SNPs) as genetic markers are widely used in population genomics, genome-wide association studies (GWAS), paternity testing, and population identification. Traditional SNP identification technologies can analyze hundreds of loci simultaneously, including TaqMan fluorescence analysis, KASPar identification technology, and direct PCR sequencing technology. The advantage is that the experimental operation is flexible when the number of SNP loci to be detected is small, but the disadvantage is that a single sample is used. When the number of SNPs to be detected is large (especially ≥50), the cost increases linearly; new detection technologies including biochips and high-throughput sequencing can realize the identification of 10 ³ -10 ⁶ SNPs, such as RAD sequencing (restriction site associated DNA sequencing) , exome sequencing and whole genome sequencing, the disadvantage is that the cost of building a library and sequencing a single sample is high, especially when the number of SNPs detected in a single sample is less than 1000, the average detection cost of a single SNP increases rapidly; when the number of SNPs to be detected in a single sample is Using multiplex PCR library construction combined with next-generation sequencing at 20-1000 can effectively reduce the average detection cost of a single SNP.

Multiplex PCR library building generally refers to adding multiple pairs of PCR primers to the PCR amplification system to amplify multiple target fragments at the same time, and then through the second step of universal primer amplification to form a nucleic acid library with adapters and sample tags required by the sequencer . Due to the addition of multiple pairs of primers at a higher concentration in the PCR amplification system, multiple primer-dimers are easily formed in multiplex PCR, and primer-dimers will directly affect the quality of the constructed library. Therefore, how to remove primer-dimers becomes a multiplex PCR construct key link in the library. According to public reports, Zuiyi Yang et al. can reduce the primer-dimer of multiplex PCR by optimizing the sequence of primer pairs. Base modification can also effectively reduce primer dimers during the reaction. Although primer-dimers can be removed by the methods disclosed above and post-PCR purification, multiplex PCR still has problems such as PCR bias caused by different efficiency of amplifying the target region and non-specific amplification caused by cross combination of primer pairs. More importantly, since the PCR amplification target regions of different primer pairs cannot overlap in liquid phase conditions, it is difficult for ordinary multiplex PCR to achieve continuous sequence analysis of long DNA fragments in a single reaction tube in a liquid phase system.

It is well known that complex living organisms are composed of many cells with specific properties, and the types and quantities of nucleic acids and proteins expressed by each cell in a specific state are different. Therefore, the detection of nucleic acid and protein indicators at the single-cell level is very important for biomedical research. have important meaning. From the perspective of detection indicators, single-cell sequencing mainly includes single-cell genome sequencing, single-cell RNA sequencing, single-cell epigenome sequencing and spatial single-cell sequencing; from the perspective of detection throughput, it is mainly divided into low-throughput single-cell sequencing (one-time detection). 1-500 cells) and high-throughput single-cell sequencing (1000-10,000 cells at a time).

High-throughput single-cell RNA detection mainly includes three implementations: water-in-oil-based droplet separation technology, microplate-based beads labeling technology, and microfluidics. Water-in-oil-based droplet segmentation technology is represented by 10X Genomics, Drop-Seq platform and inDrop platform. This technology uses microfluidic technology to encapsulate barcode-labeled microbeads and single cells in oil droplets and cleaves to release RNA containing polyA tails; each gel microbead is coupled with oligo dT containing cell tags and molecular tags. Nucleic acid sequence; mRNA is bound to the oligo dT nucleic acid molecule of cell tag and molecular tag, and then reverse transcription to cDNA from different cell sources to tag different cell tags and use for subsequent mixed library construction and sequencing analysis. Microplate-based beads labeling technologies are represented by BD CytoSeq, SeqWell and microwell-seq. This technology naturally settles cells into a microwell array with more than ten times the number of cells to ensure a single cell entry rate, and then adds cell label-labeled microbeads to the microwells to capture the mRNA after cell lysis; mRNA is bound to the cell label After the oligo dT with molecular tags, the cDNAs derived from different cells are labeled with different cell tags by reverse transcription and used for subsequent mixed library construction and sequencing analysis.

The droplet method completely isolates cells and label beads from other cells and beads through water-in-oil, effectively reducing the possibility of cross-contamination; at the same time, in addition to the realization of 3' RNA expression profiling libraries, the droplet protocol can also Tags and molecular tags are coupled with template switching sequences to achieve 5' single-cell RNA expression profiling; however, due to the instability and suspension characteristics of droplets, the single-cell library construction scheme based on droplet method cannot be used in RNA cells. The medium cannot be changed before and after labeling, thereby reducing the possibility of further complex reactions, especially the lack of positional information of the labeling beads. The microplate method avoids the problem of probabilistic collision affecting the capture efficiency in 10X, and has better cell capture efficiency; the label beads have a fixed position after falling into the microwell, and more liquid exchange operations can be performed; however, due to the The microwell is a semi-closed structure with an open top, which can cause cellular RNA to diffuse out of the well, so all current microwell methods can only construct 3' single-cell RNA expression profiling libraries.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a multi-nucleic acid co-labeled support.

Another object of the present invention is to provide a method for preparing a support for co-labeling multiple nucleic acids.

Another object of the present invention is to provide the application of multiple nucleic acid co-labeled supports.

The present invention modifies two or more nucleic acid molecules on the support, wherein one nucleic acid molecule is used to capture the target compound from the reaction pool and participate in a specific biochemical process together with other types of molecules co-labeled on the surface of the same solid-phase compound, including However, it is not limited to the application directions of multiplex PCR library construction, single-cell RNA expression profiling, single-cell transcriptome sequencing library construction, and single-cell multi-omics sequencing library construction.

Specifically, on the one hand, the present invention provides a multi-nucleic acid co-labeled support, which includes a support body and a variety of nucleic acid labels located on the surface and/or inside of the support body, nucleic acid labeled on a single support At least include: one or more first nucleic acid markers, whose role at least includes capturing specific compounds in the reaction system to the surface of the support; one or more second nucleic acid markers, whose role at least includes participating in the capture on the surface of the support. The specified biochemical reaction process for a specific compound.

According to a specific embodiment of the present invention, in the multiple nucleic acid co-labeled supports of the present invention, the support body is solid beads and/or semi-solid hydrogel beads.

According to a specific embodiment of the present invention, a plurality of nucleic acid co-labeled supports of the present invention are compositions comprising a plurality of supports.

According to a specific embodiment of the present invention, in the multiple nucleic acid co-labeled supports of the present invention, the number of the first nucleic acid label and the second nucleic acid label on the same support can be ≥ 1 and/or ≤ 10 ¹³ respectively.

According to a specific embodiment of the present invention, in the multiple nucleic acid co-labeled supports of the present invention, the sequences of multiple first nucleic acid markers on the same support are the same or different; the sequences of the first nucleic acid markers on different supports are the same or different; the sequences of multiple second nucleic acid markers on the same support are the same or different; or the sequences of the second nucleic acid markers on different supports are the same or different.

On the other hand, the present invention also provides a method for making the described multiple nucleic acid co-labeled supports, comprising:

Multiple nucleic acids are labeled on the support body by grafting and/or grafting to obtain a support with multiple nucleic acids co-labeled.

According to a specific embodiment of the present invention, the preparation method of the multiple nucleic acid co-labeled supports of the present invention includes:

The support body and the nucleic acid are respectively modified with functional units that can interact, so that the two react to label the nucleic acid on the support body;

The nucleic acid is directly synthesized on the support body according to the preset nucleotide sequence; and/or

Nucleic acid labeling is performed on the body of the support using a biochemical reaction for nucleic acid extension or ligation protocols.

On the other hand, the present invention also provides the 5' single-cell RNA expression profile analysis of the multiple nucleic acid co-labeled supports, the construction of a 5' single-cell VDJ library of a microwell array platform, and the construction of a 3' single-cell RNA library. , Construction of single-cell transcriptome library, single-cell multi-omics research, multiplex PCR and/or construction of multiplex PCR sequencing library applications.

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: template switching sequences containing cell tags and molecular tags and RNA capture sequences are fixed on the support. Specifically, at least two nucleic acid sequences are marked on the support: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence contains at least a capture sequence, which is used to capture the target nucleic acid molecule and serve as a primer for extension or reverse transcription; the second nucleic acid sequence Sequences include cell tag sequences, which are used to tag molecules derived from all mRNAs in the same cell; different kinds of supports have different cell tags. Preferably, the support is made to capture the RNA released after single cell lysis in the micropores of the chip, and the RNA derived from the same cell is labeled with the same cell label by template switching during the reverse transcription process, and then the cDNA is realized by amplification Amplified and finally constructed as a 5' single-cell RNA expression profiling library.

According to some embodiments of the invention, the multiple nucleic acid co-labeled supports of the invention are 5' single-cell VDJ libraries used to construct microwell array platforms. Wherein: template switching sequences containing cell tags and molecular tags and RNA capture sequences are fixed on the support. Specifically, at least two nucleic acid sequences are marked on the support: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence contains at least a capture sequence, which is used to capture the target nucleic acid molecule and serve as a primer for extension or reverse transcription; the second nucleic acid sequence Sequences include cell tag sequences, molecular tag sequences and template switching sequences. Cell tag sequences are used to label molecules derived from all mRNAs in the same cell; molecular tag sequences are used to label each reverse transcribed cDNA molecule from the same support. The cDNA molecules reversely transcribed from different RNAs are marked with different molecular tags; the template switching sequence can be used as a template to extend the 3' end of the reverse transcribed cDNA to mark the molecular tag sequence and cell tag sequence; different species with different cell labels on the supports. Preferably, the support is made to capture the RNA released after single cell lysis in the micropores of the chip, and in the reverse transcription process, the RNA derived from the same cell is labeled with the same cell label through template switching, and further through TCR and BCR/ The constant region primers of the Ig gene realize the enrichment of TCR and BCR/Ig nucleic acid sequences and finally break them into a high-throughput single-cell VDJ sequencing library.

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: a random primer containing a cell tag that can be conditionally blocked and an RNA capture sequence are fixed on the support, and specifically, at least two nucleic acid sequences are marked on the support: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence The sequence contains at least a capture sequence, which is used to capture the target nucleic acid molecule and serve as primer extension or reverse transcription; the second nucleic acid sequence includes a conditional blockable random primer containing a cell tag, and the cell tag sequence is used to tag all cells derived from the same cell. Molecules of mRNA; different kinds of supports have different cell tags. Preferably, the support is made to capture the RNA released after single cell lysis in the micropores of the chip and reverse transcribed into cDNA, and the subsequent random primers containing cell tags are synthesized by two strands to achieve the same tag on the cDNA derived from the same cell. Cell labeling, followed by amplification of cDNA 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 single cell transcriptome libraries. Among them: random primer sequences containing cell tags and RNA capture sequences are fixed on the support, different types of supports have different cell tags, and can detect any sequence of RNA molecules without being limited to the 3' end or 5' end; preferably, the support includes two types of supports, each type of single support has at least two nucleic acid sequences, a combination of a first nucleic acid sequence and a second nucleic acid sequence, or a third nucleic acid sequence and a third nucleic acid sequence. A combination of two nucleic acid sequences; the first nucleic acid sequence contains at least a capture sequence for capturing target nucleic acid molecules; the second nucleic acid sequence includes a random primer sequence containing a cell tag, and the cell tag sequence is used to tag 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 the lysis of single cells in the micropores of the chip, and in the process of reverse transcription, the RNA derived from the same cell is labeled with the same cell label, and then the cDNA is amplified and amplified by amplification. Finally, a single-cell RNA transcriptome library was constructed.

According to some embodiments of the present invention, the multiple nucleic acid co-labeled supports of the present invention are used in single-cell multi-omics studies. Preferably, a library for constructing RNA expression levels and/or for detecting protein expression levels by nucleic acid tags of proteins is included. Wherein: RNA capture sequences containing cell tags and capture sequences for protein-labeled nucleic acid tags are fixed on the support, and different types of supports have different cell tags. Preferably, the first nucleic acid sequence includes at least a capture sequence for capturing the target nucleic acid molecule and extending as a primer; the second nucleic acid sequence includes a cell tag sequence, a molecular tag sequence and a template switching sequence; the cell tag sequence is used to label cells derived from the same cell All mRNA molecules of the mRNA; the molecular tag sequence is used to label each reverse transcribed cDNA molecule, and the cDNA molecules reverse transcribed from different RNAs on the same support are marked with different molecular tags; the template switching sequence can be used as a The template continues to extend the 3' end of the reverse transcribed cDNA to label the molecular tag sequence and cell tag sequence; the third nucleic acid sequence includes the cell tag sequence, molecular tag sequence and protein nucleic acid tag capture sequence, and the protein nucleic acid tag capture sequence is used to capture And extend the protein nucleic acid marker in the same spatial structure as the single cell to be tested. Preferably, the support is made to capture RNA and protein nucleic acid tags released after single cell lysis in the micropores of the chip, and in the reverse transcription process, the RNA and protein nucleic acid tags derived from the same cell are labeled with the same cell tag. , and then finally constructed into a single-cell RNA transcriptome library and a protein-labeled 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 multiplex PCR sequencing libraries. Among them: the primers that can interfere with each other are respectively fixed on different supports. Specifically, the supports comprise at least two types of supports: one or more primer-labeled supports of a first kind, and one or more primer-labeled supports of a second kind, each of which is labeled with at least one A pair of nucleic acid primers: a first nucleic acid primer pair is labeled on the first type of primer-labeled support, and a second nucleic acid primer pair different from the first nucleic acid primer pair is labeled on the second type of primer-labeled support. Each kind of support can also selectively include more nucleic acid primer pairs, such as other nucleic acid primer pairs, and the target fragments amplified by multiple pairs of primer pairs on the same support do not overlap on the template; Different primer pairs can amplify different regions of interest, and these regions of interest may or may not overlap. Preferably, all the supports are mixed in proportions and then mixed with the nucleic acid template and the PCR enzyme reaction system, so as to perform a single-tube unbiased multiplex PCR.

In another aspect, the present invention also provides a kit, which includes the multiple nucleic acid co-labeled supports of the present invention. Preferably, the kit is a 5' single-cell VDJ library that can be applied to 5' single-cell RNA expression profiling, the construction of a microwell array platform, the construction of a 3' single-cell RNA library, the construction of a single-cell transcriptome library, a single-cell VDJ library Kits for multi-omics studies, multiplex PCR and/or construction of multiplex PCR sequencing libraries.

More preferably, the kit also includes one or more of the following compositions:

Composition 1: a mixture of a support containing a template switching sequence of a cell tag and a molecular tag and an RNA capture sequence, a microwell chip, a cell lysate, a reverse transcription reagent, a nucleic acid amplification reagent, and a nucleic acid interruption library building module; comprising the The kit of composition 1 can be used for 5' single-cell RNA expression profiling;

Composition 2: mixture of template switching sequences containing cell tags and molecular tags and supports for RNA capture sequences, microwell chips, cell lysates, reverse transcription reagents, constant region primers, nucleic acid amplification reagents, and nucleic acid interruption library construction Module; a kit comprising the composition 2 can be used to construct a 5' single-cell VDJ library of a microwell array platform;

Composition 3: a mixture of random primers containing cell tags and supports 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 can be used to construct 3' single-cell RNA library;

Composition 4: a support mixture containing a random primer sequence of a cell tag and an RNA capture sequence, a microwell chip, a cell lysate, a reverse transcription reagent, a two-strand synthesis module, and a nucleic acid amplification and extension reagent; a composition comprising the composition 4 The kit can be used to construct single-cell transcriptome library;

Composition 5: a capture sequence support mixture containing cell-tagged protein-tagged nucleic acids, a microwell chip, a cell lysate, a reverse transcription reagent, and a nucleic acid interrupt library building module; a kit comprising the composition 5 can be used for single-cell multiplexing. omics research;

Composition 6: premixed support mixture of coupled primers, multiplex PCR enzyme and buffer; further optionally, index primers adapted to a high-throughput sequencer; the kit comprising the composition 6 can be used for Multiplex PCR and/or construction of multiplex PCR sequencing library (pre-mixed support mixture of coupled primers and multiplex PCR enzymes and buffers, can achieve single-tube unbiased multiplex PCR; further includes adapting to high-throughput sequencers. Indexing primers to construct multiplex PCR libraries that can be used for sequencing analysis by index PCR).

To sum up, the present invention provides a support for co-labeling of multiple nucleic acids and a method for making and application thereof. The technology of the present invention can capture nucleic acid molecules on the solid surface and perform specific biochemical reactions together with other kinds of nucleic acids modified on the solid surface by carrying out a variety of nucleic acid modification schemes on solid-phase (including semi-solid) supports. . Specific types of polynucleic acid modified solid supports can be used in the fields of multiplex PCR library construction, single molecule long fragment nucleic acid sequencing library construction, single cell transcriptome sequencing library construction and single cell multi-omics sequencing library construction.

Description of drawings

1A-1C are schematic diagrams of the structures of multiple nucleic acid co-labeled supports of the present invention.

FIG. 2A and FIG. 2B are schematic diagrams of the application of multiple nucleic acid co-labeled supports of the present invention to multiplex PCR reactions.

FIG. 2C is a schematic diagram of the application of various nucleic acid-labeled supports of the present invention to the construction of multiplex PCR sequencing libraries.

FIG. 2D and FIG. 2E are schematic diagrams of the design method and structure of multiple nucleic acid co-labeling supports for multiplex PCR of the present invention.

Figure 3A is a schematic structural diagram of a multi-nucleic acid co-labeled support for 5' single-cell RNA expression profiling and the construction of a 5' single-cell VDJ library of a microwell array platform of the present invention.

Figure 3B is an experimental flow chart of the multi-nucleic acid labeling support shown in Figure 3A applied to 5' single-cell RNA expression profiling and the construction of a 5' single-cell VDJ library on a microwell array platform.

Figure 3C is a schematic diagram of the preparation of the nucleic acid co-labeling support for 5' single-cell RNA expression profiling according to the present invention.

Figure 4A is a schematic structural diagram of a multi-nucleic acid co-labeled support applied to a 3' single-cell RNA library of the present invention.

Figure 4B is a schematic flowchart of the application of the multiple nucleic acid co-labeled supports of the present invention to a 3' single-cell RNA library.

Figure 5A is a schematic structural diagram of a plurality of nucleic acid co-labeling supports for constructing a single-cell transcriptome library of the present invention.

FIG. 5B is a schematic flowchart of the use of the multiple nucleic acid co-labeled supports of the present invention to construct a single-cell transcriptome library.

FIG. 6A is a schematic structural diagram of a plurality of nucleic acid co-labeling supports for constructing a multi-omics single-cell library of the present invention.

FIG. 6B is a schematic flow chart of the multi-omics single-cell library construction using the multiple nucleic acid co-labeled supports of the present invention.

FIG. 7A shows the agarose electrophoresis results of the membrane protein nucleic acid tag sequencing library constructed by the procedure in Example 1. FIG.

Figure 7B shows the analysis results of the 5' single-cell expression profile library fragments constructed by the process in Example 1.

FIG. 7C shows the analysis results of the T cell VDJ library fragments constructed by the procedure in Example 1. FIG.

Figure 7D shows the analysis results of the B cell VDJ library fragments constructed by the procedure in Example 1.

Figure 8 shows the distribution of reads at the gene level obtained by sequencing analysis of the 3' single-cell RNA library constructed by the process in Example 2 and the single-cell transcriptome library constructed by the process in Example 3. The BD Phapsody 3' single-cell expression profile library is a library analysis structure constructed entirely with BD Rhapsody.

Figure 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 implementation process and the beneficial effects of the present invention are described in detail below through specific embodiments and examples, which are intended to help readers better understand the essence and characteristics of the present invention, and are not intended to limit the scope of implementation of this case. The methods that are not specified in the following specific embodiments and examples are carried out according to conventional operations in the field or operating conditions suggested by instrument manufacturers.

The terms "a," "an," "an," and "the" and "the" used in the description of the present invention are also intended to include plural forms, unless the context clearly dictates otherwise or the meaning of the context clearly indicates that Odd number.

The present invention first provides structures for multiple nucleic acid co-labeled supports. As shown in Figures 1A to 1C, the support body can be a solid plane (Figure 1A), a solid bead (Figure 1B) or a semi-solid hydrogel (Figure 1C); nucleic acid labels can be located on a solid surface (Figure 1A) and Figure 1B) can also be located in the loose interior of the hydrogel (Figure 1C). The nucleic acid labeled on a single support at least includes: one or more first nucleic acid labels 101, the function of which at least includes capturing a specific compound in the reaction system to the surface of the support (so the first nucleic acid label 101 is also called a capture nucleic acid label); One or more second nucleic acid labels 102, the functions of which include at least being able to participate in a specified biochemical reaction process of a specific compound captured on the surface of the support (hence the second nucleic acid label 102 is also referred to as a reactive nucleic acid label). Optionally, other types of nucleic acid labels IN may also be included on the support.

According to some specific embodiments of the present invention, a plurality of nucleic acid co-labeled supports of the present invention are compositions comprising a plurality of the above-mentioned supports (supports with the structures shown in FIG. 1A , FIG. 1B and/or FIG. 1C ) .

In a specific application, the sequences of multiple first nucleic acid markers 101 on the same support may be the same. In a specific application, the sequences of multiple first nucleic acid markers 101 on the same support may be different.

In certain applications, the sequences of the first nucleic acid markers 101 on different supports may be the same. In certain applications, the sequences of the first nucleic acid markers 101 on different supports may be different.

Under certain uses, the sequences of multiple second nucleic acid labels 102 on the same support may be identical. In certain applications, the sequences of the plurality of second nucleic acid markers 102 on the same support may be different.

In certain applications, the sequences of the second nucleic acid labels 102 on different supports may be identical. In certain applications, the sequences of the second nucleic acid labels 102 on different supports may be different.

Likewise, the sequences of multiple other types of nucleic acid markers IN on the same support may be the same or different for a specific application. The sequences of the other kinds of nucleic acid labels on different supports may be the same or different under certain applications.

According to different uses, the number of the first nucleic acid label, the second nucleic acid label, and other kinds of nucleic acid labels on the same support can be ≥ 1 and/or ≤ 10 ¹³ , respectively.

In a specific 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 function of "capturing a specific compound in the reaction system to the surface of the support" described in the present invention, or It can have the function of "participating in the specified biochemical reaction process of the specific compound captured on the surface of the support" described in the present invention. For example, such a first nucleic acid label and a second nucleic acid label may be two primers in a primer pair.

The present invention also provides a method for preparing multiple nucleic acid co-labeled supports for different purposes. Nucleic acid labeling of the support can be carried out in two ways: "graft to" and "graft from". In certain applications, nucleic acid labeling of the support can be performed using a "graft to" protocol alone. In certain applications, nucleic acid labeling of the support can be performed using the "graft from" protocol alone. Under specific applications, nucleic acid labeling of supports can be mixed using "graft to" and "graft from" protocols.

When using the "graft to" scheme, the support and nucleic acid are modified with functional units that can interact with each other, including but not limited to hydroxyl, aldehyde, epoxy, amino, carboxyl and their activated forms, phosphoric acid, alkynyl, One or more of azide, mercapto, alkene, biotin, avidin, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, tosyl ester, etc. Different types of nucleic acid labels (first nucleic acid label, second nucleic acid label, and other nucleic acid labels) can be modified with the same functional unit or with different functional units. The modified support and the nucleic acid are brought into contact under specific conditions sufficient to allow the functional units capable of interacting to react and connect to each other.

When the "graft from" scheme is adopted, it can be directly synthesized on the support according to the preset nucleotide sequence, or the nucleic acid can be labeled with the scheme of nucleic acid extension or ligation by biochemical reaction. Nucleic acid modifications well known in the art can be added to the labeled nucleic acid, including but not limited to amino, phosphate, alkynyl, azide, sulfhydryl, disulfide, alkene, biotin, azobenzene, methyl, spacer, photocleavage groups One or more of , dI, dU, LNA, XNA, ribonucleic acid bases and dideoxyribonucleic acid bases, and the like.

The present invention also provides the use of multiple nucleic acid co-labeled supports for multiplex PCR. In traditional multiplex PCR applications, all templates and primers are mixed in the same reaction system. As the target region of PCR increases, the type and total concentration of primer pairs will also increase accordingly, which is easy to form primer dimers and thus Reduce the amplification efficiency of the target region. The use of the multiple nucleic acid co-labeled supports provided by the present invention can well reduce the generation of primer dimers in multiplex PCR, and because mutual interference between PCR primers can be avoided, the continuous sequence of long fragments can be analyzed in a single tube. As shown in FIG. 2A , in this application, the multiple nucleic acid co-labeled supports provided by the present invention comprise at least two types of supports: one or more first type of primer-labeled supports 1, one or more The second kind of primer-labeled support 2, each support is labeled with at least one pair of nucleic acid primers: as shown in the figure, the first kind of primer-labeled support 1 is labeled with the first nucleic acid primer pair (No. A forward primer 201F and a first reverse primer 201R), a second nucleic acid primer pair different from the first nucleic acid primer pair (the second forward primer 202F and the second nucleic acid primer pair is labeled on the support 2 of the second kind of primers) The reverse primer 202R, the two types of supports independently can also selectively include more nucleic acid primer pairs such as other nucleic acid primer pairs (other forward primer 2NF and other reverse primer 2NR). And can be designed by PCR primer software such as Primer Primer optimizes primer sequences to reduce mutual interference between multiple primer pairs on the same magnetic bead. The target fragments amplified by multiple primer pairs on the same support do not overlap on the template. Primer pairs labeled on different supports can Different thus can amplify different target regions, and these target regions can partially overlap or not overlap. Each primer marked on the support at least includes the H region that can be combined and extended with the target region: the first forward primer H region 201FH, The first reverse primer H region 201RH, the second forward primer H region 202FH, the second forward primer H region 202RH, the other forward primer H region 2NFH, the other reverse primer H region 2NRH. Each primer marked on the above also includes at least the universal nucleic acid sequence U region: the first forward primer U region 201FU, the first reverse primer U region 201RU, the second forward primer U region 202FU, the second reverse primer U region 202RU, Other forward primer U region 2NFU, other reverse primer U region 2NRU. The forward primer U region FU or reverse primer U region RU sequence of primers on all supports in a specific use can be identical: first forward Primer U region 201FU = second forward primer U region 202FU = other forward primer U region 2NFU, first reverse primer U region 201RU = second reverse primer U region 202RU = other reverse primer U region 2NRU. The forward primer U region FU or reverse primer U region RU sequence of primers on all supports may be inconsistent. When multiplex PCR is implemented, the supports labeled with different kinds of nucleic acid primers are combined with multiplex PCR in a preset ratio. The reaction systems are mixed together, and the preset ratio is determined according to the nucleic acid amplification efficiency on different kinds of supports, which can be as low as the average ratio of different kinds of supports (for example, labeling primers P1/P2/P3 on magnetic beads to form the first type of magnetic particles). Beads, the primers P4/P5/P6 are labeled on the magnetic beads to form the second type of magnetic beads, the ratio here refers to 0.01 times the quantity ratio of the first type of magnetic beads to the second type of magnetic beads when the magnetic beads are mixed), Can be as high as 100 times the average ratio of different types of supports. Multiplex PCR reaction system to At least include DNA template, DNA polymerase, dNTP, buffer of appropriate concentration, etc. As shown in FIG. 2B , at the beginning of the multiplex PCR reaction, one of the primers marked on the support (for example, the first forward primer 201F or the second forward primer 202F) will bind to the DNA single-stranded

templates

203 and 204 in the reaction system to It is confined to the surface of the support and generates a

complementary strand

205, 206 of the DNA template under the action of a polymerase, which is subsequently dissociated from the template and is replaced by another primer on the same support (eg the first one). The reverse primer 201R, the second reverse primer 202R) are combined and extended to obtain

nucleic acid strands

207 and 208 complementary to the

complementary strands

205 and 206 of the aforementioned DNA template; this is repeated to obtain a support with a large number of nucleic acid sequences. Due to the limited types of primer pairs marked on the same support and the physical distance from primer pairs on other supports, the possibility of different primer pairs combining with each other to form dimers can be effectively reduced; Different kinds of nucleic acids can be labeled with different types of nucleic acids and the number of primer pairs on each support can be increased to effectively increase the target amplification region of multiplex PCR. For example, each support can be coupled with less than 5 primer pairs, or each support Up to 10 primer pairs can be coupled to the substrate, or up to 100 primer pairs can be coupled to each support.

Further, the present invention also provides the use of multiple nucleic acid co-labeled supports for multiplex PCR sequencing library construction. As shown in FIG. 2C , the supports with

nucleic acid sequences

207 and 208 complementary to the complementary strands of the DNA template generated in the reaction shown in FIG. 2B are used as templates for primer sequences compatible with the sequencer: the third forward primer 209F and The third reverse primer 209R performs the PCR reaction of the second step, thereby obtaining a nucleic acid sequencing library that can be used for sequencing. The primer sequences compatible with the sequencer include at least the universal binding sequence in the primers shown in Figure 2A (universal nucleic acid sequence U region, namely other forward primer U region 2NFU/other reverse primer U region 2NRU), sample label 2NFi/2NRi And the nucleic acid sequence 2NFA/2NRA compatible with the sequencer, the forward universal nucleic acid sequence U region on all supports here is the same: the first forward primer U region 201FU=the second forward primer U region 202FU=Other forward Primer U region 2NFU, reverse universal nucleic acid sequence U region on all supports is also the same: first reverse primer U region 201RU=second reverse primer U region 202RU=other reverse primer U region 2NRU. Sequencers used for sequencing include but are not limited to MGIseq sequencing platform, illumina sequencing platform, Ion sequencing platform, PacBio sequencing platform, Nanopore sequencing platform, etc.

The present invention also provides a method for making multiple nucleic acid co-labeling supports for multiplex PCR. As shown in FIG. 2D , when the continuous base sequence of the long fragment 210 needs to be sequenced and analyzed, the amplification of a single primer pair can no longer meet the needs. In this case, multiple pairs of primers need to be designed to amplify and then construct the library for sequencing. A primer pair (represented by the H region of the primer in the scheme shown in FIG. 2D, namely the first forward primer H region 201FH and the first reverse primer H region 201RH) amplifies the first target fragment 201, and uses the second primer pair ( The second forward primer H region 202FH and the second reverse primer H region 202RH) amplify the second target fragment 202, and use more primer pairs (other forward primer H region 2NFH and other reverse primer H region 2NRH) More target fragments 2N are amplified, and finally the sequencing results are spliced into the sequence of the long fragment 210 . In the traditional liquid-phase multiplex PCR reaction, since the first target fragment 201 and the second target fragment 202 partially overlap, the second forward primer and the first reverse primer together will produce small non-target amplification products, and the primers need to be Divide the multiplex PCR reaction into at least two tubes of parallel amplification, and amplify the primer pairs with no overlap of the target fragment: the first forward primer, the first reverse primer, other forward primers and other reverse primers are one tube, and the second The forward primer and the second reverse primer are in one tube. When using the multiplex PCR library building method using the multi-nucleic acid labeling support in the present invention, it can be obtained by labeling the first primer pair that amplifies the target fragment without overlapping and other primer pairs on the same magnetic bead, such as the first magnetic bead. The first type of multiple nucleic acid co-labeled supports, and the second primer pair that overlaps with the amplification target fragment of the primer pair on the first magnetic bead is labeled on another magnetic bead such as the second magnetic bead to obtain the first. Two kinds of multiple nucleic acid co-labeled supports, so that primer pairs on the two kinds of multiple nucleic acid co-labeled supports do not interfere with each other even if the primers are amplified in the same tube. According to the primer design principle shown in FIG. 2D, the present invention pre-synthesizes a

first primer pair

201F, 201R with a 5' specific modification 211, a

second primer pair

202F, 202R with a 5' specific modification 211, and more with Additional primer pairs 2NF and 2NR for 5' specific modification of 211 (Fig. 2E). 5' specific modifications include, but are not limited to, hydroxyl, aldehyde, epoxy, amino, carboxyl and their activated forms, phosphoric acid, alkynyl, azide, sulfhydryl, alkene, biotin, avidin, isothiocyanate, Isocyanates, acyl azides, sulfonyl chlorides, tosyl esters, etc., the corresponding selected supports include but are not limited to epoxy, amino, carboxyl, alkynyl, azide, alkene, heavy metal, azide, affinity and other functional groups 212. The supports with functional groups 212 are contacted and coupled with nucleic acid primers with 5' specific modifications 211 under appropriate conditions, in particular the primers capable of producing non-specific products are coupled separately on different supports, e.g. A

first primer pair

201F, 201R with a 5' specific modification 211, other primer pairs 2NF, 2NR with a 5' specific modification 211 are coupled to the first microbeads to form a first product 213, and a first product 213 will have a 5' specific modification 211 The

second primer pair

202F and 202R is coupled to the second microbead to form the second product 214 (Fig. 2E), and finally the first product 213 and the second product 214 are mixed together in proportion to form the final product with multiple nucleic acid labels. The supports are used for multiplex PCR library construction.

The present invention also provides the use of multiple nucleic acid co-labeled supports for 5' single-cell RNA expression profiling. It is well known that complex living organisms are composed of many cells with specific properties, and the types and quantities of RNAs transcribed and expressed by each cell in a specific state are different, so it is of great significance to detect RNA transcription at the single-cell level. Current technologies for detecting single-cell transcriptomes can be divided into low- and medium-throughput and high-throughput single-cell transcriptome sequencing technologies according to throughput. The medium and low-throughput single-cell transcriptome sequencing is represented by smart-seq, and the single-cell transcriptome is constructed by reverse transcription and amplification of the RNA obtained by direct lysis of a single cell; the library preparation of high-throughput single-cell transcriptome sequencing is based on oil-in-oil Represented by water microfluidics and microwell array platforms, mRNA molecules derived from different cells are reverse transcribed into corresponding mRNA molecules through oligo dT primers or template switch oligo (TSO) containing cell tags and molecular tags. Uniquely tagged cDNA molecules and further sequencing can simultaneously analyze the mRNA expression of thousands of single cells. The water-in-oil technology platform encapsulates a single cell and a single microbead containing a cell tag in a single droplet for lysis and reverse transcription in one step. According to the sequencing read near the 3' end or 5' end of the RNA, it can be divided into 3' single cells RNA expression profiling library and 5' single cell RNA expression profiling library. The microwell array platform is usually an array chip consisting of microwells with a diameter of 20-60 μM. After a single cell is lysed in the microwell, the mRNA with a polyA tail is captured by the microbeads carrying the cell-tag oligo dT primer in the same well, and then RNA reverse transcripts derived from the same cell are labeled with the same and unique cellular label by reverse transcription extension. The efficiency of the current microwell array-based single-cell sequencing library preparation platform largely depends on the RNA capture efficiency in the microwells containing oligo dT microbeads, and unlike the water-in-oil platform, the microwell array platform can only construct 3 'Single-cell RNA expression profiling library. The invention can improve the RNA capture efficiency in the micropore by carrying out various nucleic acid labels on the microbeads in the preparation of the single-cell sequencing library, and can realize the preparation of the 5' single-cell RNA expression profile library.

The present invention provides the use of a variety of nucleic acid co-labeled supports for 5' single-cell RNA expression profile analysis, and the construction of a 5'-single-cell VDJ library of a microwell array platform.

As shown in FIG. 3A , the support here is microbeads (solid microbeads or semi-solid hydrogel microbeads), 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 contains at least a capture sequence 303, which is used to capture the target nucleic acid molecule and serve as a primer for extension or reverse transcription, such as a base sequence oligo dT with a length of 15-40, which can be adjusted by adjusting the first nucleic acid sequence 301 on the support. on the quantity and density to control the efficiency of RNA capture. The first nucleic acid sequence 301 also includes a first universal sequence nucleic acid 302 and a conditionally cleavable site X under certain uses. Conditionally 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 endonuclease recognition sequences. variety.

The second nucleic acid sequence 304 is composed of one or more of the second universal nucleic acid sequence 305 , the cell tag sequence 306 , the molecular tag sequence 307 and the template switching sequence 308 . Wherein the second universal nucleic acid sequence 305 may include an adapter nucleic acid sequence that matches the sequencer, such as Read1 Sequencing Primer or Read2 Sequencing Primer in the illumina sequencer. The cell tag sequence 306 is used to tag molecules derived from all mRNAs in the same cell, with the same cell tag on each support and different cell tags on different kinds of supports. The cell tag sequence 306 can be a random or semi-random nucleic acid sequence, such as a 12bp degenerate base NNNNNNNNNNNN, or a combination of multiple fixed nucleic acid sequences, such as 96 8-base sequences and 96 8-base sequences and A random combination of 96 8-base sequences, which may or may not include connecting nucleic acid regions between the 8-base sequences. The molecular tag sequence 307 is used to label each reverse transcribed cDNA molecule, and the cDNA molecules reverse transcribed from different RNAs on the same support are marked with different molecular tags. The molecular tag 307 can 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 switching sequence 308 can be used as a template to extend the 3' end of the cDNA reverse transcribed from the first nucleic acid sequence 301 to label the molecular tag sequence 307, the cell tag sequence 306 and the second universal nucleic acid sequence 305. Template switching sequence 308 includes two or more RNA bases rG or other modified base G analogs, such as LNA or XNA, at least at the 3' end.

Figure 3B shows the experimental flow chart of the multi-nucleic acid-labeled support shown in Figure 3A in the construction of the 5' single-cell library of the microwell array platform. In this specific application, each support is coupled with two kinds of Nucleic acid markers: the first nucleic acid sequence 301 and the second nucleic acid sequence 304 . When a single support labeled with the first nucleic acid sequence 301 and the second nucleic acid sequence 304 is contacted with RNA derived from a single cell, the RNA 309 containing the complementary sequence of the second nucleic acid sequence 304 is captured by the first nucleic acid sequence 301 on the support and reversed by reverse The transcription reaction system forms a cDNA molecule 310, in which the cDNA is extended to the 5' end of the RNA 309 by the reverse transcriptase with terminal nucleotransferase function adding a continuous base C to the cDNA strand, and then the cDNA strand will be with the same support surface. The adjacent second nucleic acid sequence 304 containing more than two bases rG or its base analogs is complementary and extended to the second universal nucleic acid sequence 305 to form a complete cDNA molecule 310 with cell tags and molecular tags. Optionally, the cDNA molecule 310 can be detached from the support through the cleavable site X as a template for the next step of amplification, or can be extended through the single primer extension of the second universal nucleic acid sequence 305, which is complementary to the cDNA molecule 310. The strand is used as the template for the next amplification, or the support containing the cDNA molecule 310 after removing the first nucleic acid sequence 301 and the second nucleic acid sequence 304 not involved in the reverse transcription reaction on the support by enzymatic treatment is used as the template for the next amplification. In a subsequent step, the fragmented or unfragmented cDNA molecule 310 as a template is PCR amplified with a primer pair containing the first universal sequence 302 and the second universal sequence 305 to form a double-stranded nucleic acid 311 product. Further, double-stranded nucleic acid 311 can analyze the type and abundance of single-cell RNA expression through two library construction methods, one of which is to analyze the expression of all RNA molecules with polyA tails without bias. The library scheme is to randomly break the double-stranded nucleic acid 311, end-repair, and add a base A at the 3' end to form a molecular structure 312, which is then connected to a linker 313 containing a protruding T, and is ligated by the first primer 315 containing the first sample Index317 Amplify with the second primer 319 containing the second sample Index 321 to form the first final library 323 . The first primer 315 comprises a first nucleic acid sequence 316 compatible with the sequencer, a first sample index 317 and a sequence 318 complementary to the long-chain partial sequence in the adapter 313 . The second primer 319 includes a second nucleic acid sequence 320 compatible with the sequencer, a second sample index 321 and a sequence 322 that is identical to a partial sequence of the second universal nucleic acid sequence 305 . This library construction method can also be replaced by other random library construction schemes that can achieve the same purpose, including but not limited to the library construction scheme of transposase interrupted library construction or random primer extension. The purpose of another library construction scheme is to analyze the expression of the target gene in a targeted manner, which can be realized by two-step multiplex PCR, such as using the first gene-specific primer 324, the second gene-specific primer 326 and the universal primer 305 respectively. The primer pairs form the first-step multiplex PCR product 325 and the second-step multiplex PCR product 327, and finally use the second-step multiplex PCR product 327 as a template to pass through the first primer 315 containing the first sample Index317 and the second sample Index321 The second primer 319 amplifies to form a second final library 328. The library constructed by targeted multiplex PCR can be used for the analysis of the immune repertoire. Furthermore, the multiplex PCR product 327 in the second step can also be used to construct a full-length VDJ immune repertoire library according to the first random interrupt library construction scheme. Analysis of T cell receptor and antibody VDJ sequences. Both the first final library 323 and the second final library 328 are further used for sequencing and information analysis.

The present invention also provides a method for making a nucleic acid co-labeling support for 5' single-cell RNA expression profiling analysis. As shown in FIG. 3C, the cell tag sequence 306 consists of a first cell tag 329, a first attachment region 330, a second cell tag 331, a second attachment region 332, a third attachment region 333, and a third cell tag 334 that are sequentially linked. It consists of 6 areas. In order to co-label the first nucleic acid sequence 301 and the second nucleic acid sequence 304 on the support, the present invention pre-synthesizes the first nucleic acid sequence 301 and the third nucleic acid sequence 335 with a 5' specific modification 340. The 5' specific modification 340 includes But not limited to hydroxyl, aldehyde, epoxy, amino, carboxyl and their activated forms, phosphoric acid, alkynyl, azide, sulfhydryl, alkene, biotin, avidin, isothiocyanate, isocyanate, acyl azide , sulfonyl chloride, tosyl ester, etc., the corresponding selected supports include but are not limited to functional groups such as epoxy, amino, carboxyl, alkynyl, azide, alkene, heavy metal, azide, avidin, etc. Mission 339. The support with functional group 339 is contacted and coupled (step 341) with the first nucleic acid sequence 301 and the third nucleic acid sequence 335 with the 5' modification under appropriate conditions to form a first product 342, the first nucleic acid sequence The ratio of 301 to the third nucleic acid sequence 355 on the first product 342 can be adjusted by adding different concentrations; then the first product 342 and the fourth nucleic acid sequence 336 undergo hybridization in an environment containing an appropriate salt ion concentration, dNTP and a polymerase buffer In step 343, the second product 344 is obtained by hybridization and extension, and the fourth nucleic acid sequence 336 nucleic acid molecule sequentially contains the complementary sequence 332' of the second connecting region 332, the complementary sequence 331' of the second cell tag 331 and the first connecting region 330 from 5'. The complementary sequence 330' of the third nucleic acid sequence 335 on the support hybridizes with the complementary sequence 330' on the fourth nucleic acid sequence 336 through its own connecting region 330 sequence and extends to the sequence The second cell tag 331 and the second connecting region 332 The second product 344 is connected with the fifth nucleic acid sequence 346 under the action of DNA ligase after the complementary strand is removed under denaturing conditions to generate the third product 347; the fifth nucleic acid sequence 346 is composed of the first nucleic acid molecule 337 and The second nucleic acid molecule 338 is the double-stranded DNA obtained by annealing and hybridization in advance, the first nucleic acid molecule 337 contains the third connecting region 333, the third cell tag 334, the molecular tag sequence 307 and the template switching sequence 308 connected in sequence, and the second nucleic acid molecule 338 It contains at least the complementary sequence of the partial sequence of the second connecting region 332 and the third connecting region 333, and also includes part of the complementary sequence of the third cell tag 334 under certain conditions, so the second nucleic acid molecule 338 can be combined with the first nucleic acid molecule 337. The third connecting region 333 and the second connecting region 332 on the second product 344 are hybridized together and connected to each other by a ligase; in particular, the 5' end of the first nucleic acid molecule 337 sometimes also contains a phosphoric acid modification; finally, under denaturing conditions The third product 347 undergoes an elution step 348 to wash off the complementary nucleic acid sequence, that is, the second nucleic acid molecule 338 to form a support 349 that is finally marked with the first nucleic acid sequence 301 and the second nucleic acid sequence 304, which has the first nucleic acid sequence 301 and the second nucleic acid sequence 304. The supports 349 labeled with the two nucleic acid sequences 304 can be used directly in the library building procedure for 5' single-cell RNA expression profiling.

The present invention also provides the use of a plurality of nucleic acid co-labeled supports applied to a 3' single-cell RNA library, wherein the cell label and the reverse transcription primer oligo dT are respectively located at both ends of the cDNA molecule. Figure 4A shows at least two nucleic acid-labeled supports, where the supports are beads (solid beads or semi-solid hydrogel beads), and at least two nucleic acid sequences are labeled on the supports: the first Nucleic acid sequence 401 and second nucleic acid sequence 404.

The first nucleic acid sequence 401 contains at least a capture sequence 403, which is used to capture the target nucleic acid molecule and serve as a primer for extension or reverse transcription, such as a base sequence oligo dT with a length of 15-40, which can be adjusted by adjusting the first nucleic acid sequence 401 on the support. The number and density of the above control the efficiency of capturing RNA; the first nucleic acid sequence 401 also includes the first universal sequence nucleic acid 402 under certain uses.

The second nucleic acid sequence 404 is composed of one or more of the second universal nucleic acid sequence 405 , the cell tag sequence 406 , the primer sequence 407 and the reversible blocking site 408 . Wherein the second universal nucleic acid sequence 405 may include an adaptor nucleic acid sequence that matches the sequencer, such as Read1 Sequencing Primer or Read2 Sequencing Primer in the illumina sequencer, and the optional second universal nucleic acid sequence 405 contains a conditional breaking site X . Conditionally cleavable sites include, but are not limited to, disulfide modifications, dU modifications, RNA base modifications, dI modifications, DSpacer modifications, AP site modifications, photocleavable PC linkers, and restriction endonuclease recognition sequences. The cell tag sequence 406 is used to tag molecules derived from all mRNAs in the same cell, with the same cell tag on each support and different cell tags on different kinds of supports. The cell tag sequence 406 can be a random or semi-random nucleic acid sequence, such as a 12bp degenerate base NNNNNNNNNNNN, or a combination of multiple fixed nucleic acid sequences, such as 96 8-base sequences and 96 8-base sequences and A random combination of 96 8-base sequences, which may or may not include connecting nucleic acid regions between the 8-base sequences. The primer sequence 407 can be used as a primer to extend the cDNA molecule that is complementary to it, and can be combined and extended with the cDNA product obtained by reverse transcription of the capture sequence 403; the primer sequence 407 can be a random or semi-random nucleic acid sequence with a length of 5-15 bases, For example, 6 random degenerate bases NNNNNN, and gene-specific sequences can also be used to enrich targeted regions. The function of the reversible blocking site 408 is to prevent the non-specific extension of the primer sequence 407 as a primer when the first nucleic acid sequence 401 captures and extends the target nucleic acid, and in a specific situation, the blocking effect is released to allow the primer sequence 407 to act as a primer. extend. The reversible blocking site 408 can be simple 3' phosphate modification, ddNTP modification or C3 spacer modification, or a combination of cleavable modification and extension blocking modification, and the cleavable modification can be DSpacer modification/RNA base Modification/dU modification, etc., extension blocking modification including but not limited to LNA/XNA/3' phosphate/inverted dT/ddNTP/C3 spacer/C6 spacer/various fluorescent dyes and quenching modifications, such as reversible blocking sites 408 can be (rN)NNNN-C3 or (rN)N-C3-C3-ddN, rN represents any ribonucleotide degenerate base, N represents any deoxyribonucleotide degenerate base, C3 is an extension blocking modified C3 spacer, and ddN is a dideoxyribonucleoside; after this sequence forms a double strand with the target DNA, it can be recognized and excised by RNaseH and expose the 3' hydroxyl of primer sequence 407, thereby activating primer sequence 407 as a primer nucleic acid extension ability.

Figure 4B shows the flow chart of the experiment of constructing a 3' single-cell RNA library with the double nucleic acid labeling support shown in Figure 4A. When a single support labeled with the first nucleic acid sequence 401 and the second nucleic acid sequence 404 is contacted with RNA derived from a single cell, the RNA 409 containing the complementary sequence of the second nucleic acid sequence 404 is captured by the first nucleic acid sequence 401 on the support and reversed by reverse The transcription reaction system forms a cDNA molecule 410, and then the cDNA molecule 410 is denatured by high temperature and then melted with the RNA to be complementary to the region of the primer sequence 407 on the second nucleic acid sequence 404 near the surface of the same support; Optimally, the cDNA molecule 410 can be combined with More than one second nucleic acid sequence 404 on the surface of the support is complementary; further, the primer sequence 407 that is complementary to the cDNA molecule 410 can be recognized by a related enzyme to excise the reversible blocking site 408 and expose the 3' of the primer sequence 407 Hydroxyl, this process can be completed by RNaseH cleavage of ribonucleic acid bases, or by alkaline phosphatase alone to treat 3' phosphate, or by endonuclease that can excise AP site to form 3' hydroxyl, or by USER Enzyme\AP site excision enzyme combined with alkaline phosphatase to complete; then extended by DNA polymerase with strand replacement activity to form a first nucleic acid molecule 411 comprising a complementary sequence of first nucleic acid sequence 401 at one end and a cell tag sequence 406 at the other end; having The support of the first nucleic acid molecule 411 can be formed by a single-primer amplification method to form a complementary strand 412 and eluted from the support to amplify the template, or it can be directly cleaved from the support through the conditional cleavage site X to obtain the first nucleic acid molecule. The two nucleic acid molecules 413 are used as templates for further amplification to form a double-stranded nucleic acid product 414, wherein the forward and reverse amplification primers respectively contain all or part of the nucleic acid sequences of the first universal sequence nucleic acid 402 and the second universal sequence nucleic acid 405. Further, the double-stranded nucleic acid product 414 can be used to analyze the type and abundance of single-cell RNA expression through two library construction methods, one of which aims to unbiased analysis of the expression of all RNA molecules with polyA tails. The library construction scheme is to amplify the double-stranded nucleic acid product 414 and amplify it by the first primer 415 containing the first sample Index417 and the second primer 419 containing the second sample Index421 to form the first final library 423, wherein the first primer 415 includes The first nucleic acid sequence 416, the first sample index 417 and the first primer hybridization region 418 compatible with the sequencer, the second primer 419 includes the second nucleic acid sequence 420, the second sample index 421 and the second primer hybridization region compatible with the sequencer 422. This library construction method can also be replaced by other random library construction schemes that can achieve the same purpose, including but not limited to the library construction scheme of transposase interrupted library construction or random primer extension. The purpose of another library construction scheme is to analyze the expression of the target gene in a targeted manner, which can be achieved by two-step multiplex PCR, such as using the first gene-specific primer 424, the second gene-specific primer 426 and the universal primer 305 respectively. The primer pair is the product of two-step multiplex PCR: the product 425 of the first-step multiplex PCR and the product 427 of the second-step multiplex PCR, and finally the product 427 of the second-step multiplex PCR is used as a template to pass with the product containing the first sample Index417. The first primer 415 and the second primer 419 containing the second sample Index 421 are amplified to form a second final library 428 . Libraries constructed by targeted multiplex PCR can be used for immune repertoire analysis, especially for 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 present invention further provides the use of a multi-nucleic acid co-labeled support for constructing a single-cell transcriptome library. The cellular label can label any position of the RNA chain to form a cDNA molecule with cellular and molecular labels. As shown in Figure 5A, there are two types of support nucleic acid labeling structures, the supports here are microbeads or hydrogel beads, and there are at least two nucleic acid sequences on a single support, such as the first nucleic acid sequence 501 and the second nucleic acid sequence. The combination of the nucleic acid sequence 505, or the combination of the third nucleic acid sequence 509 and the second nucleic acid sequence 505.

The first nucleic acid sequence 501 at least contains a capture sequence 503 for capturing target nucleic acid molecules, such as a base sequence oligo dT with a length of 15-40, and the capture can be controlled by adjusting the number and density of the first nucleic acid sequence 501 on the support RNA efficiency. The first nucleic acid sequence 501 also includes a polymerase extension blocking site 504, which can prevent 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 in a specific application, and the capture efficiency of the first universal nucleic acid sequence 502 can be adjusted 503 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 matching a sequencer, such as illumina sequencing Read1 Sequencing Primer or Read2 Sequencing Primer in the instrument; cell tag sequence 507 is used to tag molecules derived from all mRNAs in the same cell, each support has the same cell tag and different types of supports have different cell tags . The cell tag sequence 507 can be a random or semi-random nucleic acid sequence, such as a 12bp degenerate base NNNNNNNNNNNN, or a combination of multiple fixed nucleic acid sequences, such as 96 kinds of 8-base sequences and 96 kinds of 8-base sequences and A random combination of 96 8-base sequences, which may or may not include connecting nucleic acid regions between the 8-base sequences. The primer sequence 508 can be combined with the RNA template as a primer extension and extended into a cDNA molecule, and can be combined with the RNA captured by the capture sequence 503 and extended. The primer sequence 508 can be a random or semi-random nucleic acid sequence with a length of 5-15 bases, For example, 6 random degenerate bases NNNNNN, and gene-specific sequences can also be used to enrich targeted regions.

The third nucleic acid sequence 509 is composed of one or more of the second universal nucleic acid sequence 506, the cell tag sequence 507, the molecular tag sequence 510 and the capture sequence 503, wherein the second universal nucleic acid sequence 506 may include an adaptor nucleic acid matching the sequencer Sequence, such as Read1 Sequencing Primer or Read2 Sequencing Primer in the illumina sequencer; the cell tag sequence 507 is used to label molecules derived from all mRNAs in the same cell, and each support has the same cell tag and different types of supports. With different cell tags, the cell tag sequence 507 can be a random or semi-random nucleic acid sequence, such as a 12bp degenerate base NNNNNNNNNNNN, or a combination of multiple fixed nucleic acid sequences, such as 96 8-base sequences and 96 A random combination of 8-base sequences and 96 kinds of 8-base sequences, the 8-base sequences may or may not include connecting nucleic acid regions; the molecular tag sequence 510 is used to label each reverse transcribed cDNA molecule, which is obtained from the same cDNA molecule. The cDNA molecules reversely transcribed from different RNAs on the support are marked with different molecular tags. 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. Base NNNNNNNNN or NNNNNNNNNV; capture sequence 503 is used to capture target nucleic acid molecules, such as base sequence oligo dT with a length of 15-40.

Figure 5B shows the experimental flow chart of the construction of single-cell transcriptome library using two types of dual nucleic acid labeling supports. When the single nucleic acid-labeled support contacts the RNA derived from a single cell, the RNA 512 containing the complementary sequence 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 RNA 512 captured on the surface of the support is Under suitable conditions, it combines with the primer sequence 508 of the second nucleic acid sequence 505 and forms a cDNA molecule 514 through a reverse transcription reaction system; 509 is the cDNA formed by the primer; the reverse transcribed support can be further digested to remove the molecules of the first nucleic acid sequence 501 , the third nucleic acid sequence 509 and the second nucleic acid sequence 505 that do not participate in the reaction. The supports containing the cDNA molecules 514 can then be analyzed for the type and abundance of single-cell RNA expression by two methods of library construction. One of the library construction methods aims to unbiased analysis of the expression of all RNA molecules with polyA tails, and this library construction scheme uses a random primer 517 extension amplification scheme. The random primer 517 is composed of a universal primer sequence 515 and a random base sequence 516: the universal primer sequence 515 can include an adaptor nucleic acid sequence matching the sequencer, such as Read2 Sequencing Primer or Read1 Sequencing Primer in the illumina sequencer; the random base sequence 516 It can be a random or semi-random nucleic acid sequence with a length of 5-15 bases, such as 9 consecutive degenerate bases NNNNNNNNN. The random primer 517 hybridizes to the cDNA molecule 514 in an appropriate environment and produces a complementary strand 518 of the cDNA molecule 514 under the action of a DNA polymerase with strand replacement activity; The primer pair of the nucleic acid sequence 506 and the universal primer sequence 515 is amplified to generate a double-stranded product 519; the double-stranded product 519 is amplified by the first primer 520 containing the first sample Index522 and the second primer 524 containing the second sample Index526 to form the first primer. A final library 528, wherein the first primer 520 comprises a sequencer-compatible first nucleic acid sequence 521, a first sample index 522 and a first primer hybridization region 523, and the second primer 524 comprises a sequencer-compatible second nucleic acid sequence 525, the second sample index526 and the partial sequence of the hybridization region 527 with the second primer; this library construction method can also be replaced with other random library construction schemes that can achieve the same purpose, including but not limited to ultrasonic interruption, enzyme interruption Or library construction schemes such as transposase interruption. The purpose of another library construction scheme is to analyze the expression of the target gene in a targeted manner, which can be achieved by two-step multiplex PCR, such as using the first gene-specific primer 529, the second gene-specific primer 531 and the universal primer 506 respectively. The primer pair is the product of two-step multiplex PCR: the product 530 of the first-step multiplex PCR and the product 532 of the second-step multiplex PCR, and finally the product 532 of the second-step multiplex PCR is used as the template to pass the first primer 520 and the second primer 520 524 is amplified to form a second final library 533. Libraries constructed by targeted multiplex PCR can be used for immune repertoire analysis, especially for full-length T cell receptor and antibody VDJ sequences. Both the first final library 528 and the second final library 533 were further used for sequencing and information analysis.

The present invention also provides the use of multiple nucleic acid co-labeled supports for single-cell multi-omics research. According to the central dogma of biology, DNA carrying genetic information transmits the information to RNA through transcription and is translated into protein, which finally performs the main biological function. However, due to the complexity of the physiological system, the expression levels of RNA and protein are not consistent, and RNA cannot directly reflect the post-translational modification and interaction of proteins. Therefore, it is very important to study the expression of RNA and protein in the same cell at the same time. 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 here 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 the third nucleic acid sequence 609.

The first nucleic acid sequence 601 contains at least a capture sequence 603 for capturing the target nucleic acid molecule and extending as a primer, such as a base sequence oligo dT with a length of 15-40, which can be adjusted by adjusting the number and amount of the first nucleic acid sequence 601 on the support. Density to control the efficiency of capturing RNA; under specific use, the first nucleic acid sequence 601 also includes the first universal nucleic acid sequence 602 and a conditional cleavage site X, and the conditionally cleavable site includes but is 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 the second universal nucleic acid sequence 605, the cell tag sequence 606, the molecular tag sequence 607 and the template switching sequence 608, wherein the second universal nucleic acid sequence 605 may include a sequencer matching sequence Adapter nucleic acid sequence, such as Read1 Sequencing Primer or Read2 Sequencing Primer in illumina sequencer; cell tag sequence 606 is used to label molecules derived from all mRNAs in the same cell, each support has the same cell tag and different types of support There are different cell tags on the object, and the cell tag sequence 606 can be a random or semi-random nucleic acid sequence, such as 12bp degenerate base NNNNNNNNNNNN, or it can be a combination of multiple fixed nucleic acid sequences, such as 96 kinds of 8-base sequences With the random combination of 96 kinds of 8-base sequences and 96 kinds of 8-base sequences, the connecting nucleic acid region may or may not be included between the 8-base sequences; the molecular tag sequence 607 is used to label each reverse transcribed cDNA molecule, The cDNA molecules reverse transcribed from different RNAs on the same support are marked with different molecular tags. 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 and base NNNNNNNNN or NNNNNNNNNV; the template switching sequence 608 can be used 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 cell tag sequence 606 and the second universal nucleic acid sequence 605 , the template switching 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 is composed of one or more of the third universal nucleic acid sequence 610, the cell tag sequence 606, the molecular tag sequence 607 and the protein nucleic acid tag capture sequence 611, wherein the cell tag sequence 606 and the molecular tag sequence 607 are the same as the first The structures of the two nucleic acid sequences 604 are consistent; the third universal nucleic acid sequence 610 is inconsistent with the second universal nucleic acid sequence 605 and contains an adaptor nucleic acid sequence that matches the sequencer, such as Read1 Sequencing Primer or Read2 Sequencing Primer in the illumina sequencer; protein The nucleic acid tag capture sequence 611 is used to capture and extend the protein nucleic acid tag in the same spatial structure as the single cell to be tested. 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 the cell where the cell is located. chamber or droplet.

FIG. 6B shows the experimental flow chart of the construction of a multi-omics single-cell library with the three nucleic acid labeling supports shown in FIG. 6A . First, the cells to be tested are contacted and bound with a nucleic acid-labeled antibody molecule 612 that recognizes a specific protein in advance, and the non-specifically bound nucleic acid-conjugated antibody is washed away. The structure of the antibody molecule 612 includes complementary binding to the protein nucleic acid tag capture sequence 611 The sequence 613, the protein-specific sequence 614, the fourth universal primer sequence 615, and the molecule 616, the fourth universal primer sequence 615 is inconsistent with the second universal nucleic acid sequence 605, the third universal nucleic acid sequence 610 and contains a matching adapter with the sequencer Nucleic acid sequences, such as Read2 Sequencing Primer or Read1 Sequencing Primer in the illumina sequencer, molecule 616 refers to specific antibodies in this process, and can also be small molecular compounds, carbohydrates, peptides and other substances that bind to the target detection protein. When a single support is contacted with a single cell bound with nucleic acid-conjugated antibody molecules 612, cell lysis releases RNA 617 and nucleic acid-conjugated antibody molecules 612 and is separated by the first nucleic acid sequence 601 and the third nucleic acid sequence 609 on the support, respectively Capture and form a cDNA molecule 618 or a DNA molecule 619 through a reverse transcription reaction system, wherein the cDNA is extended to the 5' end of RNA617 by a reverse transcriptase with terminal nucleotransferase function to add a continuous base C to the cDNA strand, and then the The cDNA strand will be complementary to the template switching sequence 608 containing two or more bases rG or its base analogs near the surface of the same support and continue to extend to the second universal nucleic acid sequence 605 region to form a complete cell tag and molecular tag. cDNA molecule 618; alternatively, the cDNA molecule 618 and the DNA molecule 619 can be detached from the support through the cleavable site X as a template for the next step of amplification, or can pass the second universal nucleic acid sequence 605 and the fourth universal nucleic acid sequence 605. The extended chain complementary to the cDNA molecule 618 or the DNA molecule 619 formed after the single primer extension of the nucleic acid sequence 615 is used as a template for the next amplification, or the first nucleic acid sequence 601, the first nucleic acid sequence 601, the first nucleic acid sequence 601, the first nucleic acid sequence 601 on the support that does not participate in the reverse transcription reaction are removed by enzymatic treatment. After the second nucleic acid sequence 604 and the third nucleic acid sequence 609, the support containing the

cDNA molecules

618 and 619 is used as the template for the next step of amplification; in the subsequent step, the mixture of the fragmented or unfragmented cDNA molecule 618 and the DNA molecule 619 is used as the template A first double-stranded nucleic acid product 621 and a second double-stranded nucleic acid product are formed by PCR amplification with the first universal nucleic acid sequence 602/second universal nucleic acid sequence 605 and the third universal nucleic acid sequence 610/fourth universal nucleic acid sequence 615 double primer pair 620 mixture. Further, the formed double-stranded nucleic acid product can be used for single-cell multi-omics analysis through three library construction methods. The purpose of the first library construction scheme is to construct a nucleic acid library that can analyze the abundance of the protein to be detected, and the first library 630 can be obtained directly by PCR amplification of the first index primer 622 and the second index primer 626; wherein the first index primer 622 includes The sequencer-compatible first nucleic acid sequence 623, the first sample index 624 and the nucleic acid sequence 625 complementary to the fourth universal nucleic acid sequence 615 are sequentially linked, and the second index primer 626 includes the sequencer-compatible first sequencer The second nucleic acid sequence 627, the second sample index 628 and the nucleic acid sequence 629 complementary to the third universal nucleic acid sequence 610 are composed. The purpose of the second library construction scheme is to analyze the expression of all RNA molecules with polyA tails without bias. This library construction scheme is to randomly interrupt the mixture of the first double-stranded nucleic acid product and the second double-stranded nucleic acid product. End repair and add base A at the 3' end to form a molecular structure 631, which is then ligated with a linker 632 containing an overhang T and amplified by the first primer 634 containing the first sample Index624 and the second primer 636 containing the second sample index628 Augmentation forms a second final library 638, wherein the first primer 634 comprises a sequencer-compatible first nucleic acid sequence 623, a first sample index 624, and a nucleic acid sequence 635 complementary to the first universal nucleic acid sequence 602, connected in sequence, and the second The primer 636 includes a sequencer-compatible second nucleic acid sequence 627, a second sample index 628 and a nucleic acid sequence 637 complementary to the second universal nucleic acid sequence 605, which can also be replaced by other methods that can achieve the same purpose. Random library construction schemes, including but not limited to transposase interrupt library construction or random primer extension library construction schemes. The purpose of the third library construction scheme is to analyze the expression of the target gene in a targeted manner, which can be achieved by two-step multiplex PCR, such as using the first gene-specific primer 639 and the second gene-specific primer 641 and the second universal primer respectively. The primer pair composed of 605 forms a multiplex PCR product: the first step multiplex PCR product 640 and the second step multiplex PCR product 642, and finally the second step multiplex PCR product 642 is used as a template to pass through with the first primer 634 containing the first sample Index624. A third final library 643 is formed by amplifying with the second primer 636 containing the second sample Index 628 . The library constructed by targeted multiplex PCR can be used for the analysis of the immune repertoire. Furthermore, the multiplex PCR product 642 in the second step can also be used to construct a full-length VDJ immune repertoire library according to the first random interrupt library construction scheme. Analysis of T cell receptor and antibody VDJ sequences. The library 630, the second final library 638, and the third final library 643 are all further used for sequencing and information analysis.

Embodiment 1: the support of multiple nucleic acid co-labeling is applied to 5' single-cell RNA expression profile library construction and VDJ library construction and multi-omics library construction

In this example, a variety of nucleic acid co-labeled supports were prepared according to the following operation steps and applied to construct a 5' single-cell RNA expression profile library, a VDJ library and a multi-omics library.

1 Make a variety of nucleic acid-labeled magnetic beads

1.1 Synthesize the single-stranded nucleic acid of the following sequence.

1.2 At the concentration of 0.25M EDC, 384 kinds of 300pmol amino-modified CB1 single-stranded nucleic acid (SEQ ID No.2) and 300pmol amino-modified dT single-stranded nucleic acid (SEQ ID No.1) and 60,000 30μM carboxyl magnetic beads were respectively mixed Rotate and mix at room temperature for 3 hours, and after washing twice, 384 nucleic acid-labeled magnetic beads were obtained.

1.3 Mix the 384 nucleic acid-labeled magnetic beads obtained in 1.2 and evenly distribute them into a 384-well plate.

1.4 The single-stranded nucleic acid CB2-TSO with the same Cell Barcode (SEQ ID No.3, whose 3 '(rG)'s are expressed as the G of RNA) together with CB2-T7 (SEQ ID No.5) and rCB (SEQ ID No. 4) annealed to form a double-stranded structure with sticky ends, in which the n'n'n'n'n'n'n'n'CTGTAG sequence in rCB2 was identical to that in CB2-TSO and CB2-T7. CTACAGnnnnnnnn is a reverse complementary sequence; nnnnnnnn is an 8bp Cell barcode sequence, and there are 384 types in this embodiment.

1.5 Add 384 kinds of annealed CB2-rCB2 double-stranded nucleic acids to a 384-well plate containing magnetic beads in the following proportions, and react at 22°C for 30 minutes.

试剂reagent	50μL体系50μL system
2X Rapid Ligation Buffer2X Rapid Ligation Buffer	25μL25μL
CB2-rCB2双链核酸CB2-rCB2 double-stranded nucleic acid	3μL3μL
T4 DNA LigaseT4 DNA Ligase	3μL3μL
RNase-free waterRNase-free water	补充至50μLMake up to 50 μL

1.6 After the reaction is completed, mix 384 kinds of magnetic beads, remove the complementary strands by high temperature treatment at 95°C, and store at 4°C for later use.

2. Cell labeling of single-cell cDNA and protein using polynucleic acid-labeled magnetic beads

2.1 Incubate the antibody against human CD4 molecule conjugated with T7 nucleic acid sequence with fresh PBMC cells to make the antibody fully bind to the CD4 protein on the cell membrane surface, and then wash with fresh PBS.

2.2 Process the chip according to the microwell chip instructions provided in the BD Rhapsody Single Cell Sequencing Kit, and add 10,000 incubated PBMC cells.

2.3 Add 300,000 magnetic beads prepared in step 1 to the microplate, and wash off the excess magnetic beads after magnetic suction holes.

2.4 Add the lysis solution that comes with the kit, and after 2 minutes, magnetically suck out the magnetic beads and wash them.

2.5 Prepare 200uL of reverse transcription reagent according to the following reaction and suspend the magnetic beads.

试剂reagent	200μL体系200μL system
Superscript II first-strand buffer(5×)Superscript II first-strand buffer(5×)	4040
DTT(100mM)DTT (100mM)	1010
Betaine(5M)Betaine(5M)	4040
MgCl2(1M)MgCl2 (1M)	1.21.2
dNTP 10mM dNTP 10mM	2020
RNAse inhibitor RNAse inhibitor	55
SuperScript II reverse transcriptaseSuperScript II reverse transcriptase	1010
RNase-free waterRNase-free water	73.873.8

2.6 Reverse transcription according to the following conditions.

2.7 Directly remove the reverse transcription supernatant, add the following 200uL exonuclease reaction solution, and react at 37°C for 30 minutes to remove excess primers on the magnetic beads.

试剂reagent	200μL体系200μL system
10X外切酶缓冲液10X Exonuclease Buffer	20μL20μL
外切酶exonuclease	10μL10μL
RNase-free waterRNase-free water	170μL170μL

3 Construction of high-throughput sequencing library of single-cell membrane protein expression

3.1 Wash the magnetic beads with the elution buffer that comes with the BD Rhapsody kit and resuspend them. After treating at a high temperature of 95°C for 5 min, aspirate the supernatant immediately for use; the remaining magnetic beads are resuspended in the elution buffer for use.

3.2 Configure the following PCR Mix.

组分component	For 1 library(μL)For 1 library(μL)
PCR MasterMix(Cat.No.91-1118)PCR MasterMix (Cat.No.91-1118)	100100
Universal Oligo(Cat.No.650000074)Universal Oligo(Cat.No.650000074)	1010

Bead RT/PCR Enhancer(Cat.No.91-1082)Bead RT/PCR Enhancer(Cat.No.91-1082)	1212
Primer T7(SEQ ID No.7)Primer T7 (SEQ ID No.7)	1010
2.2.8中洗脱上清Elution supernatant in 2.2.8	6868
Total Total	200200

3.3 Purify with SPRI beads 1.4× after amplification according to the following conditions, and elute with 30uL.

3.4 Configure the following Index PCR mix.

PCR MasterMix(Cat.No.91-1118)PCR MasterMix (Cat.No.91-1118)	2525
Index P5 primer(SEQ ID No.9)Index P5 primer (SEQ ID No.9)	22
Index P7 primer(SEQ ID No.10)Index P7 primer (SEQ ID No. 10)	22
Nuclease-free waterNuclease-free water	1818
2.3.3中的洗脱液Eluent in 2.3.3	33
Total Total	5050

3.5 After amplification according to the following conditions, use SPRI beads 0.8× to purify, and wash with 30uL of sterile water to obtain the membrane protein CD4 single-cell expression library. The library fragment size is about 280bp, which meets the library inspection standard, as shown in Figure 7A.

4 Construction of single cell 5' expression profile library and immune receptor VDJ library

4.1 Full-length cDNA amplification: configure the following PCR reaction system and resuspend the magnetic beads prepared in 2.3.1.

Kit组分Kit Components	For 1 library(μL)For 1 library(μL)
PCR MasterMix(Cat.No.91-1118)PCR MasterMix (Cat.No.91-1118)	6060
Universal Oligo(Cat.No.650000074)Universal Oligo(Cat.No.650000074)	1010
Primer Full(SEQ ID No.8)Primer Full(SEQ ID No.8)	1010
RNase-free waterRNase-free water	4040
TotalTotal	120120

4.2 Amplify according to the following conditions and use SPRI beads 0.6× to purify, and 30uL of elution is used for later use.

4.3 Use the 5'Library Construction Kit (PN-1000020) in the Chromium Single Cell V(D)J Reagent Kits of 10×genomics company to interrupt and construct the library to obtain a 5' single cell expression profile library. , as shown in Figure 7B, it can be seen from the figure that the main peak of the constructed library is around 484, which meets the library inspection standard.

4.4 Use the Enrichment Kit (Human T Cell, PN-1000005 and Human B Cell, PN-1000016) in the Chromium Single Cell V(D)J Reagent Kits of 10×genomics company to amplify and label the eluted product in 4.2. The library was constructed to obtain single-cell VDJ libraries of T cells and B cells, as shown in Figure 7C and Figure 7D, respectively. It can be seen from Figure 7C that the size of the constructed library is between 200-1000 bp, which is in line with expectations. It can be seen from Figure 7D that the main peak of the constructed library is around 545, which meets the library inspection standard.

Example 2: Multiple nucleic acid co-labeled supports applied to 3' single-cell RNA library construction

In this example, a variety of nucleic acid co-labeled supports were prepared according to the following operation steps and applied to construct a 3' single-cell RNA library.

1 Make a variety of nucleic acid-labeled magnetic beads

1.1 Synthesize the single-stranded nucleic acid of the following sequence.

1.2 384 kinds of 300pmol amino-modified CB1 single-stranded nucleic acid (SEQ ID No. 2) and 300 pmol amino-modified SP2-dT30VN single-stranded nucleic acid (SEQ ID No. 11) and 60,000 30 μM carboxyl groups were prepared at a concentration of 0.25M EDC. The magnetic beads were rotated and mixed at room temperature for 3 hours, and 384 nucleic acid-labeled magnetic beads were obtained after washing twice.

1.4 Single-stranded nucleic acid CB2-NrNx (SEQ ID No. 12, wherein "(rN)" is expressed as RNA base, and the 3' end of the sequence is modified with C3) and rCB2 are annealed to form a double-stranded with sticky ends The structure, in which the n'n'n'n'n'n'n'CTGTAG sequence in rCB2 and the CTACAGnnnnnnnn in CB2-NrNx are the reverse complementary sequences; nnnnnnnn is the 8bp Cell barcode sequence, with a total of 384 types.

1.5 Add 384 kinds of annealed CB2-NrNx/rCB2 double-stranded nucleic acids to a 384-well plate containing magnetic beads in the following proportions, and react at 22°C for 30 minutes.

试剂reagent	50μL体系50μL system
2X Rapid Ligation Buffer2X Rapid Ligation Buffer	25μL25μL
CB2-NrNx/rCB2双链核酸CB2-NrNx/rCB2 double-stranded nucleic acid	3μL3μL
T4 DNA LigaseT4 DNA Ligase	3μL3μL
RNase-free waterRNase-free water	补充至50μLMake up to 50 μL

2 Use polynucleic acid-labeled magnetic beads for 3' single-cell RNA library construction

2.1 Peripheral blood was drawn and fresh PBMC cells were obtained and resuspended in PBS.

2.2 Process the chip according to the microwell chip instructions provided in the BD Rhapsody single-cell library construction kit, and add 10,000 incubated PBMC cells.

2.3 Add 300,000 magnetic beads prepared in step 2.1 to the microplate, and wash off the excess magnetic beads after magnetic suction.

2.5 Follow the instructions in the BD Rhapsody Single Cell Library Construction Kit to reverse transcription and use ExoI to excise unused primers on the magnetic beads.

2.6 Random primer NrNx double-strand extension: configure the following hybridization reaction system and suspend the magnetic beads obtained in 3.2.5.

Kit组分Kit Components	For 1 library(μL)For 1 library(μL)
WTA Extension Buffer(Cat.No.91-1114)WTA Extension Buffer(Cat.No.91-1114)	2020
Nuclease-free water(Cat.No.650000076)Nuclease-free water(Cat.No.650000076)	150150
RNase HIIRNase HII	44
TotalTotal	174174

2.7 Hybridize according to the following temperature conditions.

2.8 Add the following extension reagents.

Kit组分Kit Components	For 1 library(μL)For 1 library(μL)
10mM dNTP(Cat.No.650000077)10mM dNTP (Cat.No.650000077)	88
Bead RT/PCR Enhancer(Cat.No.91-1082)Bead RT/PCR Enhancer(Cat.No.91-1082)	1212
WTA Extension Enzyme(Cat.No.91-1117)WTA Extension Enzyme(Cat.No.91-1117)	66
Total Total	2626

2.9 Extend according to the conditions specified by the BD kit (the following conditions).

2.10 Wash the magnetic beads after extension, configure the following reaction system and suspend the magnetic beads.

Kit组分Kit Components	For 1 library(μL)For 1 library(μL)
PCR MasterMix(Cat.No.91-1118)PCR MasterMix (Cat.No.91-1118)	6060
Universal Oligo(Cat.No.650000074)Universal Oligo(Cat.No.650000074)	1010
UPP-2(SEQ ID No.13)UPP-2 (SEQ ID No. 13)	1010
RNase-free waterRNase-free water	4040

2.11 Carry out PCR according to the following reaction conditions, then use SPRI beads 0.9× to purify and elute with 30uL.

2.12 Configure the following Index PCR mix.

PCR MasterMix(Cat.No.91-1118)PCR MasterMix (Cat.No.91-1118)	2525
Index P5 primerIndex P5 primer	22
Index P7 primerIndex P7 primer	22
Nuclease-free waterNuclease-free water	1111
3.2.11中的洗脱液Eluent in 3.2.11	1010
Total Total	5050

2.13 Amplify according to the following conditions and use SPRI beads 0.5/0.25× sorting, eluting with 30uL sterile water to obtain 3’ single-cell RNA library.

2.14 Illumina NovaSeq 6000 sequenced and analyzed the position of the detected nucleic acid sequence in the RNA. As shown in Figure 8, the detected nucleic acid sequence was mainly located near the 3' end of the RNA.

Example 3: Multiple nucleic acid co-labeled supports for single-cell transcriptome library construction

In this example, multiple nucleic acid co-labeled supports were prepared according to the following operation steps and applied to construct a single-cell transcriptome library.

1 Make a variety of nucleic acid-labeled magnetic beads

1.1 Synthesize the single-stranded nucleic acid of the following sequence.

SEQ ID No.SEQ ID No.	名称name	序列sequence
1414	CB2-dN6CB2-dN6	CTACAGnnnnnnnnNNNNNNCTACAGnnnnnnnnNNNNNN

1.4 Annealing the single-stranded nucleic acid CB2-dN6 (SEQ ID No. 14) and rCB2 to form a double-stranded structure with sticky ends, wherein the n'n'n'n'n'n'n'CTGTAG sequence in rCB2 and CB2- CTACAGnnnnnnnn in dN6 is a reverse complementary sequence; nnnnnnnn is an 8bp Cell barcode sequence, with a total of 384 types.

1.5 Add 384 kinds of annealed CB2-dN6/rCB2 double-stranded nucleic acids to a 384-well plate containing magnetic beads in the following proportions, and react at 22°C for 30 minutes.

试剂reagent	50μL体系50μL system
2X Rapid Ligation Buffer2X Rapid Ligation Buffer	25μL25μL
CB2-dN6/rCB2双链核酸CB2-dN6/rCB2 double-stranded nucleic acid	3μL3μL
T4 DNA LigaseT4 DNA Ligase	3μL3μL
RNase-free waterRNase-free water	补充至50μLMake up to 50 μL

2 Single-cell transcriptome library construction using polynucleic acid-labeled magnetic beads

2.5 Follow the instructions in the BD Rhapsody single-cell library construction kit to reverse transcription and use ExoI to excise unused primers on the magnetic beads. The reverse transcription reaction conditions are modified as follows:

步骤step	温度temperature	时间 time		转速Rotating speed
11	常温normal temperature	30min30min	普通旋转normal rotation
22	37℃37℃	30min30min	1200rpm1200rpm

2.6 The following steps are carried out in strict accordance with BD Rhapsody mRNA Whole Transcriptome Analysis (WTA) Library Preparation Protocol to carry out hybridization extension and amplification to form the final library.

2.7 Illumina NovaSeq 6000 sequencing, and analyze the position of the detected nucleic acid sequence in the RNA.

The nucleic acid sequences measured by this method are more evenly distributed over the full length of RNA than BD rhapsody and the library in Example 2. As shown in Figure 8, for 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 distribution of reads at the gene level was obtained through sequencing analysis. The BD Phapsody 3' single-cell expression profile library is a library analysis structure constructed entirely with BD Rhapsody. As can be seen from the figure, the sequences contained in the 3' single-cell RNA library constructed in Example 2 are mainly distributed at the 3' end of the gene, while the sequences contained in the single-cell transcriptome library constructed in Example 3 are significantly higher than The 3' single-cell RNA library and the BD Phapsody 3' single-cell expression profiling library were more biased towards the middle of the gene.

Example 4: Multiple nucleic acid co-labeled supports for multiplex PCR sequencing library construction

The purpose of this example is to realize single-tube multiplex PCR detection of the full-length gene sequences of Brca1 and Brca2, and the design of multiplex PCR primers is 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 to SEQ ID No.348.

1 Extraction of genomic DNA

Extract 200uL of human peripheral blood genomic DNA according to the instructions of Tiangen blood genomic DNA extraction kit, and use Qubit to measure the concentration.

2 Making polynucleic acid labeled magnetic beads

2.1 Synthesize 5' amino-modified primer sequences according to the sequences in Table 1, and mix together equal amounts of primers with the same pool number.

2.2 The primer mixtures (Pool 1, Pool 2, Pool 3, Pool 4, Pool 5, Pool 6, Pool 7, Pool 8, Pool 9, Pool 10, 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) were mixed with 10 mg of magnetic beads (Dynabeads MyOne Carboxylic Acid) for 3 hours at room temperature, and washed twice to obtain 17 types of nucleic acid-labeled magnetic beads.

2.3 Mix the obtained 17 types of nucleic acid-labeled magnetic beads according to the ratio of 1:1 before use.

3 Multiplex PCR Amplification

3.1 Configure the PCR system according to the following table.

试剂reagent	50μL体系50μL system
2x QIAGEN Multiplex PCR Master Mix2x QIAGEN Multiplex PCR Master Mix	25μL25μL
核酸标记磁珠Nucleic acid labeled magnetic beads	5μL5μL
基因组DNAgenomic DNA	10ng10ng
RNase-free waterRNase-free water	补充至50μLMake up to 50 μL

3.2 Follow the procedure below on the PCR machine.

4 Index PCR to form a library

4.1 Configure the index PCR system according to the following table.

试剂reagent	50μL体系50μL system
2x KAPA Hifi2x KAPA Hifi	25μL25μL
I5 PrimerI5 Primer	5μL5μL
I7 PrimerI7 Primer	5μL5μL
RNase-free waterRNase-free water	补充至50μLMake up to 50 μL

4.2 Use the configured 50μL index PCR system to suspend the washed magnetic beads in 1.3.2, and run the following PCR program:

4.3 Use SPRI beads to purify DNA in the range of 300-500bp in length, measure the concentration and use Caliper for fragment length analysis, as shown in Figure 9, to obtain a library with a main peak of about 379bp, which meets the library inspection standard.

Claims

A kind of multi-nucleic acid co-labeled support, it includes the support body and a variety of nucleic acid labels located on the surface and/or inside of the support body, the nucleic acid labeled on a single support at least includes: one or more first nucleic acid labels , whose role at least includes capturing a specific compound in the reaction system to the surface of the support; one or more second nucleic acid labels, whose role at least includes participating in the specified biochemical reaction process of the specific compound captured on the surface of the support.
The multiple nucleic acid co-labeled supports according to claim 1, wherein the support body is solid beads and/or semi-solid hydrogel beads.
The multi-nucleic acid co-labeled support of claim 1, which is a composition comprising a plurality of supports.
The multiple nucleic acid co-labeled supports according to claim 3, wherein the number of the first nucleic acid label and the second nucleic acid label on the same support can be ≥ 1 and/or ≤ 10 13 respectively.
The multiple nucleic acid co-labeled support according to claim 3, wherein,

The sequences of multiple first nucleic acid markers on the same support are the same or different;

The sequences of the first nucleic acid markers on different supports are the same or different;

The sequences of multiple second nucleic acid labels on the same support are the same or different; or

The sequences of the second nucleic acid labels on the different supports are the same or different.
The method for making a multi-nucleic acid co-labeled support according to any one of claims 1 to 5, the method comprising:

Multiple nucleic acids are labeled on the support body by grafting and/or grafting to obtain a support with multiple nucleic acids co-labeled.
The manufacturing method according to claim 6, comprising:

The support body and the nucleic acid are respectively modified with functional units that can interact, so that the two react to label the nucleic acid on the support body;

The nucleic acid is directly synthesized on the support body according to the preset nucleotide sequence; and/or

Nucleic acid labeling is carried out on the support body using a biochemical reaction for nucleic acid extension or ligation protocols.
The multiple nucleic acid co-labeled supports according to any one of claims 1 to 5 are used for 5' single cell RNA expression profile analysis, construction of a 5' single cell VDJ library of a microwell array platform, construction of a 3' single cell RNA library, Applications in the construction of single-cell transcriptome libraries, single-cell multi-omics studies, multiplex PCR and/or construction of multiplex PCR sequencing libraries.
The application according to claim 8, wherein the multiple nucleic acid co-labeled supports are used for 5' single-cell RNA expression profiling: wherein:

A template switching sequence containing a cell tag and a molecular tag and an RNA capture sequence are immobilized on the support. Specifically, at least two nucleic acid sequences are marked on the support: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence at least contains The capture sequence is used to capture the target nucleic acid molecule and serve as a primer for extension or reverse transcription; the second nucleic acid sequence includes a cell tag sequence, and the cell tag sequence is used to label molecules derived from all mRNAs in the same cell; different kinds of supports have different cell labels;

Preferably, the support is made to capture the RNA released after single cell lysis in the micropores of the chip, and the RNA derived from the same cell is labeled with the same cell label by template switching during the reverse transcription process, and then the cDNA is realized by amplification Amplified and finally constructed as a 5' single-cell RNA expression profiling library.
The application according to claim 8, wherein, the multi-nucleic acid co-labeled support is a 5' single-cell VDJ library for constructing a microwell array platform; wherein:

A template switching sequence containing a cell tag and a molecular tag and an RNA capture sequence are immobilized on the support. Specifically, at least two nucleic acid sequences are marked on the support: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence at least contains The capture sequence is used to capture the target nucleic acid molecule and serve as a primer for extension or reverse transcription; the second nucleic acid sequence includes a cell tag sequence, a molecular tag sequence and a template switching sequence, and the cell tag sequence is used to label molecules derived from all mRNAs in the same cell ; Molecular tag sequences are used to label 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 reverse transcribed The 3' end of the cDNA continues to be extended to be labeled with molecular tag sequences and cell tag sequences; different kinds of supports have different cell tags;

Preferably, the support is made to capture the RNA released after single cell lysis in the micropores of the chip, and in the reverse transcription process, the RNA derived from the same cell is labeled with the same cell label through template switching, and further through TCR and BCR/ The constant region primers of the Ig gene realize the enrichment of TCR and BCR/Ig nucleic acid sequences and finally break them into a high-throughput single-cell VDJ sequencing library.
The application according to claim 8, wherein the multiple nucleic acid co-labeled supports are used to construct a 3' single-cell RNA library; wherein:

Conditionally blocked random primers containing cell tags and RNA capture sequences are immobilized on the support. Specifically, at least two nucleic acid sequences are marked on the support: a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence is at least Contains a capture sequence for capturing the nucleic acid molecule of interest and serves as primer extension or reverse transcription; the second nucleic acid sequence includes a conditionally blockable random primer containing a cell tag, which is used to label all mRNAs derived from the same cell. Molecules; different cell tags on different kinds of supports;

Preferably, the support is made to capture the RNA released after single cell lysis in the micropores of the chip and reverse transcribed into cDNA, and the subsequent random primers containing cell tags are synthesized by two strands to achieve the same tag on the cDNA derived from the same cell. Cell labeling, followed by amplification of cDNA to construct a 3' single-cell RNA library.
The application according to claim 8, wherein the multiple nucleic acid co-labeled supports are used to construct a single-cell transcriptome library; wherein:

Random primer sequences containing cell tags and RNA capture sequences are fixed on the support. Different types of supports have different cell tags, which can detect any sequence of RNA molecules without being limited to the 3' end or 5' end; Preferably, the support comprises two types of supports, each type of support having at least two nucleic acid sequences on a single support, a combination of a first nucleic acid sequence and a second nucleic acid sequence, or a third nucleic acid sequence and a second nucleic acid A combination of sequences; the first nucleic acid sequence comprises at least a capture sequence for capturing target nucleic acid molecules; the second nucleic acid sequence comprises a random primer sequence containing a cell tag, and the cell tag sequence is used to label molecules derived from all mRNAs in the same cell; Three nucleic acid sequences include cell tag sequences and capture sequences;

Preferably, the support is made to capture the RNA released after the lysis of single cells in the micropores of the chip, and in the process of reverse transcription, the RNA derived from the same cell is labeled with the same cell label, and then the cDNA is amplified and amplified by amplification. Finally, a single-cell RNA transcriptome library was constructed.
The application according to claim 8, wherein the multiple nucleic acid co-labeled supports are used for single-cell multi-omics research; preferably, including a library for constructing RNA expression levels and/or by protein Nucleic acid tags are used to detect protein expression levels; where:

An RNA capture sequence containing a cell tag and a capture sequence for a nucleic acid tag for labeling proteins are immobilized on the support, and different types of supports have different cell tags; preferably, the first nucleic acid sequence at least contains a capture sequence for The target nucleic acid molecule is captured and extended as a primer; the second nucleic acid sequence includes a cell tag sequence, a molecular tag sequence and a template switching sequence; the cell tag sequence is used to tag molecules derived from all mRNAs in the same cell; the molecular tag sequence is used to tag each Each reverse-transcribed cDNA molecule, the cDNA molecules reverse-transcribed from different RNAs on the same support are marked with different molecular tags; the template switching sequence can be used as a template to continue to extend the 3' end of the reverse-transcribed cDNA to Molecular tag sequence and cell tag sequence are labeled; the third nucleic acid sequence includes cell tag sequence, molecular tag sequence and protein nucleic acid tag capture sequence. The protein nucleic acid tag capture sequence is used to capture and extend the protein in the same spatial structure as the single cell to be tested. Nucleic acid markers;

Preferably, the support is made to capture RNA and protein nucleic acid tags released after single cell lysis in the micropores of the chip, and in the reverse transcription process, the RNA and protein nucleic acid tags derived from the same cell are labeled with the same cell tag. , and then finally constructed into a single-cell RNA transcriptome library and a protein-labeled nucleic acid library through amplification.
The application according to claim 8, wherein the multiple nucleic acid co-labeled supports are used to construct a multiplex PCR sequencing library; wherein:

The primers capable of interfering with each other are respectively immobilized on different supports; specifically, the supports comprise at least two kinds of supports: supports labeled with one or more primers of the first kind, and supports labeled with one or more primers of the second kind. Labeled supports, each support is labeled with at least one pair of nucleic acid primers: the first nucleic acid primer pair is labeled on the first type of primer-labeled support, and the second kind of primer-labeled support is labeled with a pair of nucleic acid primers. The first nucleic acid primer pair is different from the second nucleic acid primer pair, and each of the two types of supports can optionally include more nucleic acid primer pairs, such as other nucleic acid primer pairs, amplified by multiple pairs of primer pairs on the same support. The target fragments do not overlap on the template; the primer pairs marked on different supports are different, so that different target regions can be amplified, and these target regions can be partially overlapped or not overlapped;

Preferably, all the supports are mixed in proportions and then mixed with the nucleic acid template and the PCR enzyme reaction system, so as to perform a single-tube unbiased multiplex PCR.
A kit comprising the multiple nucleic acid co-labeled supports according to any one of claims 1 to 5;

Preferably, the kit is a 5' single-cell VDJ library that can be applied to 5' single-cell RNA expression profiling, the construction of a microwell array platform, the construction of a 3' single-cell RNA library, the construction of a single-cell transcriptome library, a single-cell VDJ library Kits for multi-omics studies, multiplex PCR and/or construction of multiplex PCR sequencing libraries;

More preferably, the kit also includes one or more of the following compositions:

Composition 1: a mixture containing a template switching sequence of a cell tag and a molecular tag and a support for an RNA capture sequence, a microwell chip, a cell lysate, a reverse transcription reagent, a nucleic acid amplification reagent, and a nucleic acid interruption library building module;

Composition 2: mixture of template switching sequences containing cell tags and molecular tags and supports for RNA capture sequences, microwell chips, cell lysates, reverse transcription reagents, constant region primers, nucleic acid amplification reagents, and nucleic acid interruption library construction module;

Composition 3: a mixture of random primers containing cell tags and supports for RNA capture sequences, a microwell chip, a cell lysate, a reverse transcription reagent, a two-strand synthesis module, and a nucleic acid amplification and extension reagent;

Composition 4: a support mixture containing a random primer sequence of a cell tag and an RNA capture sequence, a microwell chip, a cell lysate, a reverse transcription reagent, a two-strand synthesis module, and a nucleic acid amplification and extension reagent;

Composition 5: a capture sequence support mixture containing a cell-tagged protein-tagged nucleic acid, a microwell chip, a cell lysate, a reverse transcription reagent, and a nucleic acid interrupt library building module;

Composition 6: premixed primer-coupled support mixture, multiplex PCR enzyme and buffer; further optionally, index primers adapted to a high-throughput sequencer are included.