CN115896958A

CN115896958A - Gene library construction method, library construction kit, device and readable storage medium

Info

Publication number: CN115896958A
Application number: CN202211703283.7A
Authority: CN
Inventors: 毛雅超; 万成; 马淑杰; 任军; 陆思嘉
Original assignee: Yikon Genomics Shanghai Co ltd
Current assignee: Yikon Genomics Shanghai Co ltd
Priority date: 2022-12-28
Filing date: 2022-12-28
Publication date: 2023-04-04

Abstract

The application discloses a gene library construction method, a library construction kit, equipment and a readable storage medium, wherein the method comprises the following steps: cracking cells to be detected to obtain a cracking product; the cleavage product has gDNA and mRNA which are cleaved to release the cells to be detected; adding the cleavage product into a reverse transcription reaction mixed solution with a reverse transcription primer to carry out reverse transcription reaction, and synthesizing cDNA corresponding to mRNA; based on the cDNA and gDNA, a gene library of the cells to be tested is constructed. In the application, the total transcriptome of the cell to be detected and the library of the genome can be simultaneously obtained in a single experiment, the single cell genome and the transcriptome can be simultaneously researched, an effective tool is provided for researching the relation among the copy number variation, the gene mutation and the phenotypic variation of the chromosome, and a premise is provided for disclosing more cell characteristics.

Description

Gene library construction method, library construction kit, device and readable storage medium

Technical Field

The application relates to the technical field of bioinformatics, in particular to a gene library construction method, a library construction kit, a device and a readable storage medium.

Background

Single cell-related research has become the current focus of research, and DNA whole genome sequencing and RNA transcriptome sequencing-related technologies for single cells have rapidly progressed in recent years. Among them, the Amplification method of single cell genomic DNA is a method of generating PicoPLEX and Multiple Amplification Cycles (MALBAC) successively from the first Multiple Displacement Amplification (MDA); meanwhile, the single-cell mRNA amplification also generates Smart-Seq, CEL-Seq, drop-Seq and other new technologies in sequence, greatly improves the reverse transcription-amplification efficiency of trace mRNA in a single cell, and realizes the single-cell transcriptome sequencing.

However, single cell samples have many technical problems with the sensitivity and preference for isolating and amplifying nucleic acids from single cells due to the contained nucleic acids and trace amounts thereof. At present, most of the research of single cell technology can only be used for sequencing genome DNA or transcriptome sequencing of RNA, the analysis methods can not display the complete appearance of single cells, only one-dimensional data can be obtained in one experiment, and particularly, for rare and trace samples, the research speed is obviously inhibited by research means. Therefore, how to obtain the genome and the transcriptome of a single cell simultaneously in a single experiment, obtain the genome copy number information and the transcriptome information in the single cell, perform comprehensive analysis, and display the state of the cell in an all-round and multi-layer manner remains a difficult point of research.

Although some technologies for sequencing the genome of a single cell and the transcriptome together exist at present, the technologies are complex to operate, expensive in cost, high in requirements on experimental environment and difficult to realize large-scale application.

In summary, how to effectively solve the problems of simultaneously studying genome and transcriptome of a single cell in a single implementation is a technical problem that needs to be solved by those skilled in the art.

Disclosure of Invention

The application aims to provide a gene library construction method, a library construction kit, equipment and a readable storage medium, which can simultaneously research a single cell genome and a transcriptome in one experiment, provide an effective tool for researching the relation among copy number variation, gene mutation and phenotypic variation of chromosomes and provide a premise for disclosing more cell characteristics.

In order to solve the technical problem, the application provides the following technical scheme:

a method of constructing a gene library, comprising:

cracking cells to be detected to obtain a cracking product; the cleavage product has gDNA and mRNA which are cleaved to release the cell to be detected;

adding the cleavage product into a reverse transcription reaction mixed solution with a reverse transcription primer to carry out a reverse transcription reaction, and synthesizing cDNA corresponding to the mRNA;

and constructing a gene library of the cell to be detected based on the cDNA and the gDNA.

Optionally, adding the cleavage product into a reverse transcription reaction mixture with a reverse transcription primer to perform a reverse transcription reaction, and synthesizing cDNA corresponding to the mRNA, including:

adding the cleavage product into the reverse transcription reaction mixed solution for reverse transcription reaction to synthesize a cDNA first chain; wherein the poly (T) of the reverse transcription primer is capable of specific complementary paired binding to a poly (A) tail on the mRNA;

and (3) utilizing a template conversion primer to guide and synthesize a second cDNA chain by taking the first cDNA chain as a template under the polymerization activity of reverse transcriptase.

Optionally, the method for synthesizing the second strand of the cDNA using the first strand of the cDNA as a template under the polymerization activity of reverse transcriptase using a template switch primer comprises:

adding a specified number of nucleotides C to the 3' end of the first strand cDNA, and using the template switch primer to guide synthesis of the second strand cDNA under the polymerization activity of the reverse transcriptase; wherein the 3' end of the template switching primer has a G of LNA matching the specified number.

Optionally, after synthesizing the cDNA corresponding to the mRNA, before constructing the gene library of the test cell based on the cDNA and the gDNA, the method further comprises:

purifying to obtain a purified product; the purified product includes the cDNA and the gDNA.

Optionally, constructing a gene library of the test cell based on the cDNA and the gDNA, comprising:

and simultaneously amplifying the cDNA and the gDNA, and introducing a sequencing universal primer and a tag sequence to obtain a library corresponding to the total transcriptome and genome of the cell to be detected.

Optionally, after constructing the gene library of the test cell, the method further comprises:

sequencing the gene library.

Optionally, simultaneously amplifying said cDNA and said gDNA, comprising:

and sequentially carrying out linear pre-amplification reaction and exponential amplification reaction by taking the cDNA second chain and the gDNA as templates.

Optionally, performing a linear pre-amplification reaction comprising:

performing linear pre-amplification reaction in a pre-amplification reaction mixed solution by using the cDNA second chain and the gDNA as templates; wherein the pre-amplification reaction mixture comprises: a pre-amplification primer, a nucleotide monomer mixture, a nucleic acid polymerase and a primer having a complementary sequence or the same sequence as part or all of the sequencing universal primer; wherein the pre-amplification primer comprises a universal sequence and a variable sequence from a 5 'end to a 3' end.

Optionally, performing an exponential amplification reaction comprising:

adding the exponential amplification reaction mixed solution into a linear pre-amplification reaction product to perform exponential amplification reaction; wherein the exponential amplification reaction mixture comprises: an exponential amplification primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the exponential amplification primer comprises a specific sequence and a universal sequence from a 5 'end to a 3' end.

A banking kit comprising: the kit comprises a kit body and a library building reagent stored in the kit body;

the library establishing reagent comprises reverse transcriptase, reverse transcription reaction mixed liquor with a reverse transcription primer and a template conversion primer;

wherein the poly (T) of the reverse transcription primer can be specifically and complementarily paired and combined with a poly (A) tail on mRNA in total RNA of a cell to be detected;

the 3' end of the template switching primer has G of LNA matching with the specified number;

based on the library construction kit, the steps of the gene library construction method are realized.

Optionally, the banking reagent further comprises: at least one of a surfactant, a lyase, and an inhibitor that inhibits RNA degradation.

An electronic device, comprising:

a memory for storing a computer program;

a processor for implementing the steps of the gene library construction method when the computer program is executed.

A readable storage medium, on which a computer program is stored, which computer program, when executed by a processor, implements the steps of the above-described gene library construction method.

The method provided by the embodiment of the application is applied to crack the cell to be detected to obtain a cracked product; the cleavage product has gDNA and mRNA which are cleaved to release the cells to be detected; adding the cleavage product into a reverse transcription reaction mixed solution with a reverse transcription primer to carry out reverse transcription reaction, and synthesizing cDNA corresponding to mRNA; based on the cDNA and gDNA, a gene library of the cells to be tested is constructed.

In the present application, after the test cells are lysed, the total RNA and gDNA of the test cells can be released. Because the reverse transcription reaction mixture has a reverse transcription primer, the reverse transcription reaction of mRNA takes place under the action of the reverse transcription primer, and cDNA is synthesized. Thus, based on the cDNA and gDNA, a gene library of test cells can be constructed. Therefore, in the application, the total transcriptome of the cell to be detected and the library of the genome can be obtained simultaneously in a single experiment, the single cell genome and the transcriptome can be researched simultaneously, an effective tool is provided for researching the relation among the copy number variation, the gene mutation and the phenotypic variation of the chromosome, and a premise is provided for disclosing more cell characteristics.

Correspondingly, the embodiment of the application also provides a library construction kit, a device and a readable storage medium corresponding to the gene library construction method, which have the technical effects and are not described herein again.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments or related technologies of the present application, the drawings needed to be used in the description of the embodiments or related technologies are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a flow chart showing an embodiment of a method for constructing a gene library according to the present invention;

FIG. 2 is a diagram of an mRNA and reverse transcription primer in an embodiment of the present application;

FIG. 3 is a schematic diagram of a first strand of a synthesized cDNA according to an example of the present application;

FIG. 4 is a schematic diagram of template conversion addition of a first strand of cDNA in an example of the present application;

FIG. 5 is a schematic diagram of the second strand synthesis process a of cDNA in the examples of the present application;

FIG. 6 is a schematic diagram of second strand synthesis process b of cDNA in the examples of the present application;

FIG. 7 is an electrophoretogram according to an embodiment of the present application;

FIG. 8 is a graph showing experimental results in the examples of the present application;

fig. 9 is a schematic structural diagram of an electronic device in an embodiment of the present application;

fig. 10 is a schematic structural diagram of an electronic device in an embodiment of the present application.

Detailed Description

In order that those skilled in the art will better understand the disclosure, the following detailed description is given with reference to the accompanying drawings. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort belong to the protection scope of the present application.

Referring to FIG. 1, FIG. 1 is a flow chart of a gene library construction method in the present application, which can be implemented based on the library construction kit provided in the present application, and the method includes the following steps:

s101, cracking the cell to be detected to obtain a cracking product.

Wherein the cleavage product contains gDNA and mRNA which are cleaved to release the cells to be detected.

Wherein, the cell to be detected can be a single cell or a trace cell. The specific organisms to which the cells belong are not limited in this embodiment.

Specifically, the cell to be detected may be lysed by a common lysis method such as thermal lysis, which is not described herein again.

Lysis releases the total RNA (Ribonucleic Acid) and gDNA (DNA, i.e., genomic deoxyribonucleic Acid, as opposed to extrachromosomal DNA (e.g., plasmids)) of the test cell.

S102, adding the cleavage product into a reverse transcription reaction mixed solution with a reverse transcription primer to perform a reverse transcription reaction, and synthesizing cDNA corresponding to mRNA.

Adding the cleavage product into a reverse transcription translation mixed solution with a reverse transcription primer, and carrying out a reverse transcription reaction on mRNA in the cleavage product so as to synthesize cDNA corresponding to the mRNA.

In one embodiment of the present application, the step of adding the cleavage product to a reverse transcription reaction mixture with a reverse transcription primer to perform a reverse transcription reaction to synthesize cDNA corresponding to mRNA comprises:

step one, adding a cleavage product into a reverse transcription reaction mixed solution for reverse transcription reaction to synthesize a cDNA first chain; wherein the poly (T) of the reverse transcription primer is capable of specific complementary paired binding to a poly (A) tail on the mRNA;

and step two, using the template conversion primer to guide and synthesize a second cDNA chain by taking the first cDNA chain as a template under the polymerization activity of reverse transcriptase.

For convenience of description, the above two steps will be described in combination.

And adding the cleavage product into a reverse transcription reaction mixed solution with a reverse transcription primer to perform a reverse transcription reaction, and synthesizing a first cDNA chain. cDNA (complementary DNA or copy DNA) refers to a DNA strand complementary to RNA after reverse transcription in vitro.

Wherein the poly (T) of the reverse transcription primer can be specifically and complementarily paired and combined with the poly (A) tail on the mRNA in the total RNA of the cell to be detected.

In the embodiment of the present application, the reverse transcription primer can be designed in advance, so that the poly (T) of the reverse transcription primer can be specifically and complementarily paired and combined with the poly (a) tail on the mRNA (Messenger RNA, also called Messenger RNA) in the total RNA of the cell to be tested.

Since the poly (A) tail of mRNA in total RNA binds to the poly (T) specific complementary pair of specifically designed reverse transcription primers and a reverse transcription reaction is performed in the reverse transcription reaction mixture, the first strand of cDNA (herein this first strand is referred to as the first strand of cDNA) is synthesized, for example, in FIGS. 2 to 3. The gDNA released by cell lysis does not bind to the poly (T) -containing specially designed reverse transcription primer and therefore reverse transcription does not occur.

Poly (dT) may comprise 25 to 30T bases.

Specifically, the method for synthesizing the second strand of cDNA under the polymerization activity of reverse transcriptase by using the first strand of cDNA as a template through a template switching primer comprises the following steps: adding a specified number of nucleotides C at the 3' end of the first strand of cDNA, and using a template to switch primers, and under the polymerization activity of reverse transcriptase, leading to synthesize a second strand of cDNA; wherein the 3 'end of the template switch primer has G's of LNA matching the specified number.

That is, a prescribed number of nucleotides C are added to the 3' end of the first strand of cDNA, and the second strand of cDNA is synthesized under the polymerization activity of reverse transcriptase by using a template switch primer.

Wherein the 3 'end of the template switch primer has G's of LNA matching the specified number. The LNA (Locked Nucleic Acid, an oligonucleotide derivative) structure has a rigid structure formed by the 2'-O and 4' -C positions of beta-D-ribofuranose through the shrinkage, and LNA nucleotides comprise six bases of A, C, G, T, U and mC.

A template switching primer having G of LNA matching a specified number at the 3' end thereof may be designed in advance.

The specified number may be specifically 3, or 2, 4, or 5, and may be set and selected according to actual needs, which is not limited herein. Next, the synthesis of the second strand cDNA will be described by taking the designated number as 3 as an example.

That is, about 3 nucleotides C can be added to the 3' -end of the first strand of cDNA synthesized by reverse transcription without a template by a template switching reaction (as shown in FIG. 4). The 3 'end of the specially designed template switching primer contains 3 LNA G, and the 3 LNA G of the specially designed primer can be combined with about 3 nucleotides C added to the 3' end of the first strand cDNA without template, and can guide the second strand cDNA synthesis under the polymerization activity of reverse transcriptase (see FIG. 5-FIG. 6).

Wherein the reverse transcriptase is selected from the group consisting of: invitrogen ^TM SuperScript ^TM II Reverse Transcriptase, or Invitrogen ^TM SuperScript ^TM III Reverse Transcriptase, or Thermo Scientific ^TM Maxima H Minus Reverse Transcriptase, or Thermo Scientific ^TM RevertAid Reverse Transcriptase。

It should be noted that the gDNA released by cell lysis does not undergo template switching and therefore does not interfere with the second strand cDNA (i.e., second strand cDNA) synthesis reaction.

S103, constructing a gene library of the cells to be detected based on the cDNA and the gDNA.

In the present embodiment, the gene library includes a genomic library and a transcriptome library of the test cells. Specifically, a plurality of DNA fragments containing different genes of a certain organism are introduced into a population of recipient bacteria and stored, and each recipient bacteria contains different genes of the organism, which are called gene libraries. If the library contains all the genes of an organism, the gene library is called a genomic library. If the library contains only a portion of a gene from an organism, the gene library is called a partial gene library, such as a cDNA library, and mRNA is obtained first and then reverse transcribed to obtain cDNA, forming the library. The cDNA library is different from the genome library in that components such as introns and the like are removed in the mRNA splicing process, so that the cDNA library can be directly used for DNA recombination.

In the present application, after the test cells are lysed, the total RNA and gDNA of the test cells can be released. Because the reverse transcription reaction mixture has a reverse transcription primer, the reverse transcription reaction of mRNA takes place under the action of the reverse transcription primer, and cDNA is synthesized. Thus, based on the cDNA and gDNA, a gene library of a test cell can be constructed. Therefore, in the application, the total transcriptome of the cell to be detected and the library of the genome can be obtained simultaneously in a single experiment, the single cell genome and the transcriptome can be researched simultaneously, an effective tool is provided for researching the relation among the copy number variation, the gene mutation and the phenotypic variation of the chromosome, and a premise is provided for disclosing more cell characteristics.

It should be noted that, based on the above embodiments, the embodiments of the present application also provide corresponding improvements. In the preferred/improved embodiment, the same steps as those in the above embodiment or corresponding steps may be referred to each other, and corresponding advantageous effects may also be referred to each other, which are not described in detail in the preferred/improved embodiment herein.

In one embodiment of the present application, after synthesizing cDNA corresponding to mRNA, before constructing a gene library of a cell to be tested based on cDNA and gDNA, purification treatment may be performed to obtain a purified product; the purified product includes cDNA and gDNA.

That is, after second strand synthesis of cDNA, purification may be performed, thereby obtaining a purified product. The purified product includes gDNA and cDNA released during cell lysis.

In this embodiment, how to purify the product to obtain a purified product may refer to related purification operations, which are not described herein again.

In one embodiment of the present application, a gene library of test cells is constructed based on cDNA and gDNA, comprising:

Specifically, the simultaneous amplification of cDNA and gDNA comprises:

and (3) taking the second strand of the cDNA and the gDNA as templates, and sequentially carrying out linear pre-amplification reaction and exponential amplification reaction.

Wherein performing a linear pre-amplification reaction comprises:

taking the second strand of cDNA and gDNA as templates, and carrying out linear pre-amplification reaction in a pre-amplification reaction mixed solution; wherein the pre-amplification reaction mixed solution comprises: a pre-amplification primer, a nucleotide monomer mixture, a nucleic acid polymerase and a universal primer which has a complementary sequence or the same sequence with part or all of sequencing; wherein the pre-amplification primer comprises a universal sequence and a variable sequence from the 5 'end to the 3' end.

Wherein performing an exponential amplification reaction comprises:

adding the exponential amplification reaction mixed solution into a linear pre-amplification reaction product to perform exponential amplification reaction; wherein, the mixed solution of the exponential amplification reaction comprises: the kit comprises an exponential amplification primer, a nucleotide monomer mixture and a nucleic acid polymerase, wherein the exponential amplification primer comprises a specific sequence and a universal sequence from a 5 'end to a 3' end.

Specifically, cDNA and gDNA are simultaneously amplified, and universal primers and tag sequences for illumina Sequencing are introduced at the time, so that a library applicable to a Next-Generation Sequencing (NGS) platform is formed while signals of the cDNA and the gDNA are amplified by using a very small amount of cDNA and gDNA as templates.

Simultaneously amplifying cDNA and gDNA, comprising: and (3) taking the second strand of the cDNA and the gDNA as templates, and sequentially carrying out linear pre-amplification reaction and exponential amplification reaction.

Wherein performing a linear pre-amplification reaction comprises: taking the second cDNA chain and gDNA as templates, and carrying out linear pre-amplification reaction in a pre-amplification reaction mixed solution;

wherein the pre-amplification reaction mixed solution comprises: a pre-amplification primer, a nucleotide monomer mixture, and a nucleic acid polymerase;

wherein the pre-amplification primer comprises a universal sequence and a variable sequence from the 5 'end to the 3' end.

Wherein the pre-amplification reaction mixed solution comprises: has a complementary sequence or the same sequence as part or all of the sequencing universal primer.

Performing an exponential amplification reaction comprising: adding the exponential amplification reaction mixed solution into a linear pre-amplification reaction product to perform exponential amplification reaction;

wherein, the mixed solution of the exponential amplification reaction comprises: an exponential amplification primer, a nucleotide monomer mixture, and a nucleic acid polymerase;

wherein, the exponential amplification primer comprises a specific sequence and a universal sequence from the 5 'end to the 3' end.

That is, the second strand of cDNA and gDNA synthesized can be used as templates to complete a linear pre-amplification reaction in the pre-amplification reaction mixture; wherein the pre-amplification reaction mixture comprises: pre-amplification primers, nucleotide monomer mixture, and nucleic acid polymerase. The pre-amplification primers include universal sequences and variable sequences from the 5 'end to the 3' end.

By way of example: the nucleic acid polymerase may be selected from: phi29DNA polymerase, bst DNA polymerase, pyrophage3137. Vent polymerization 10 enzyme, TOPO Taq DNA polymerase, 9 ^° Nm polymerase, klenow fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7phaseDNA polymerase variant, ultra-fidelity DNA polymerase, taq polymerase, bst DNA polymerase, E.coli DNA polymerase, longAmpTaq DNA polymerase, oneTaq DNA polymerase, deepVent DNA polymerase, vent (exo-) DNA polymerase, deep Vent (exo-) DNA polymerase, and any combination thereof.

Then, using the synthesized pre-amplification product as a template, in an exponential amplification reaction mixture, further amplifying gDNA and cDNA signals for a limited number of cycles, and simultaneously obtaining a library which can be used for sequencing. Wherein, the mixed solution of the exponential amplification reaction comprises: an exponential amplification primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the exponential amplification primer comprises or consists of a specific sequence and a universal sequence from 5 'end to 3' end. Specifically, the exponential amplification primers include sequences that are complementary or identical to part or all of the sequencing primers.

Specifically, the reagent for the simultaneous amplification of gDNA and cDNA may comprise one or more components selected from the group consisting of: nucleotide monomer mixtures (e.g., dATP, dGTP, dTTP and dCTP, e.g., at a total concentration of between 1mmol and 8 mmol/. Mu.L), dTT (e.g., at a concentration of between 1mmol and 7 mmol/. Mu.L), mg2+ solutions (e.g., at a concentration of between 2mmol and 8 mmol/. Mu.L), bovine Serum Albumin (BSA), pH adjusters (e.g., tris HCl), DNase inhibitors, RNase, SO42-, cl-, K +, ca2+, na +, and/or (NH 4) +.

In one embodiment of the present application, after constructing the gene library of the test cell, the gene library can be sequenced.

That is, after obtaining a gene library corresponding to the total transcriptome and genome of the test cell, the gene library may be sequenced. The obtained gene library comprises a transcriptome library and a genome library, so that after sequencing, the data of the transcriptome and the genome can be simultaneously obtained, and then analysis can be carried out in two omics dimensions. Specifically, for how to sequence the library, a related sequencing implementation process may be referred to, and details are not repeated here.

In order to facilitate the skilled person to better understand the method for constructing a gene library provided in the embodiments of the present application, the method for constructing a gene library will be described in detail below by taking the test cell as a single cell.

As can be seen from the above, the present application can utilize mRNA released after cell lysis to reverse-transcribe and construct a library with the released gDNA. The specific implementation process comprises the following steps:

step 1, cracking single cells, and releasing gDNA and RNA;

step 2, reverse transcribing the mRNA in the mixed solution of the reverse transcription reaction by using a reverse transcription joint primer under the action of reverse transcriptase to obtain a first chain of the cDNA, wherein the RNA is obtained in the step 1; the number of cycles ranged from 5 to 10, with the number of cycles in this example being 8.

Step 3, adding about 3 nucleotides C at the 3' end of a first chain of cDNA synthesized by reverse transcription without a template;

step 4, synthesizing a second chain of the cDNA by taking the synthesized first chain of the cDNA as a template under the polymerization activity of reverse transcriptase; the number of cycles ranged from 9 to 12, with 10 in this example.

Wherein, the steps 2 to 4 are a first temperature cycle program.

Specifically, the first temperature cycle program includes: a temperature program that enables mRNA to bind a specifically designed primer to a DNA single-stranded template under the mediation of the primer; a temperature program capable of extending the length of a first type of primer bound to a DNA single-stranded template under the action of a nucleic acid polymerase to produce a pre-amplification product; the process is repeated for a specified first number of cycles.

Wherein the specified first number of cycles is greater than 1.

Step 5, taking the synthesized second strand of cDNA and gDNA as templates, and completing a linear pre-amplification reaction in a pre-amplification reaction mixed solution; wherein the pre-amplification reaction mixture comprises: pre-amplification primers, nucleotide monomer mixture, and nucleic acid polymerase. The pre-amplification primer comprises a universal sequence and a variable sequence from a 5 'end to a 3' end;

step 6, taking the synthesized pre-amplification product as a template, further amplifying gDNA and cDNA signals through limited cycle number in an exponential amplification reaction mixed solution, and simultaneously obtaining a library for sequencing; wherein the exponential amplification reaction mixture comprises: an exponential amplification primer, a nucleotide monomer mixture and a nucleic acid polymerase, wherein the exponential amplification primer comprises or consists of specific sequences and universal sequences from 5 'end to 3' end. In particular, the exponential amplification primers may comprise sequences that are complementary or identical to part or all of the sequencing primers.

The verification proves that the application can release total RNA and gDNA after thermal cracking of 3-2000 cells in about 6 hours as a template, reverse transcription is carried out on mRNA under the action of reverse transcriptase under the condition of not influencing the gDNA to obtain a first cDNA chain, and then a second cDNA chain is formed after template displacement reaction. Then, carrying out pre-amplification and exponential amplification reaction by taking double-stranded cDNA and genomic gDNA as templates, and adding an illumina library joint and a tag sequence at two ends of the cDNA and the gDNA so as to obtain a high-quality sequencing library.

The method is simple, convenient and quick to operate, can finish reverse transcription of mRNA in a short time to form cDNA, and can simultaneously amplify the cDNA and gDNA to construct a sequencing library. The method can obtain over 95 percent of reverse transcription and amplification library construction success rate, cDNA and gDNA libraries can be seamlessly jointed with mainstream sequencing platforms such as illumina or Huada BGI, off-line data can detect over 60 percent of gene expression, and chromosome copy number change with over 10Mb resolution can be detected. The sample size required to be input can be as low as 3 cells.

Corresponding to the above method embodiments, the present application also provides a library construction kit, and the library construction kit described below and the gene library construction method described above can be referred to each other.

The library building kit (herein referred to as kit for short) comprises: the kit comprises a kit body and a library building reagent stored in the kit body;

wherein, the poly (T) of the reverse transcription primer can be specifically and complementarily paired and combined with the poly (A) tail on the mRNA in the total RNA of the cell to be detected;

the 3 'end of the template switching primer has G's of LNA matching the specified number;

Further, the library establishing reagent also comprises: at least one of a surfactant, a lyase, and an inhibitor that inhibits degradation of RNA.

The kit further comprises components capable of lysing the cells. For example, one or more surfactants (e.g., NP-40, tween, SDS, tritonX-100, EDTA, guanidinium isothiocyanate), and/or one or more lyases (e.g., proteinase K, pepsin, papain).

That is, components capable of lysing cells may be included in the kit. For example, one or more surfactants (e.g., NP-40, tween, SDS, tritonX-100, EDTA, guanidinium isothiocyanate), and/or one or more lytic enzymes (e.g., proteinase K, pepsin, papain), and Invitrogen to inhibit RNA degradation ^TM SUPERase·In ^TM RNase inhibitor。

In order to facilitate the technical understanding of the implementation process and the technical effects of the gene library constructing method provided in the embodiments of the present application, the following detailed description will be given with reference to specific experiments.

The specific implementation process comprises the following steps:

step 1, obtaining single cells or trace cells:

(1) and cell dilution: PMBC separated from fresh blood of human (female with normal chromosome copy number used in this example) was diluted with PBS buffer, centrifuged, resuspended, and prepared into cell suspension, and single cells were picked up under a 10 × microscope using a mouth pipette;

(2) and placing the single cells into a PCR tube containing 4 mu L of cell lysis buffer, and picking 20, 10, 5 and 3cells respectively as experimental samples. The volume of the PBS solution containing the single cell sample must not exceed 1 μ L, and the reaction volume is strictly controlled to facilitate reverse transcription.

(3) Placing 15 x n μ L of RT Buffer in a PCR tube (n is the number of reactions);

samples and RT buffer were heat treated in a pre-heated PCR instrument with the conditions shown in Table 1.

TABLE 1

Temperature of	Time
		72℃	3min
Immediately placed on ice (0 ℃ C.)	>3min

Step 2: obtaining double-stranded full-length cDNA by reverse transcription:

taking 2.5 mu L of reverse transcriptase into the thermally treated RTbuffer in the last step, uniformly mixing in a vortex mode, and centrifuging;

step (2), to each cell lysate, the mixture of 17.5. Mu. LRT buffer and reverse transcriptase of the previous step was added, immediately after the flash separation, placed on ice, and the samples were incubated on a PCR machine under the conditions of Table 2.

TABLE 2

And 3, step 3: the method comprises the following steps: add 40. Mu.L of PCR mix into the reverse transcription product of the previous step, mix well and centrifuge, and react in a PCR instrument under the conditions shown in Table 3.

TABLE 3

And 4, step 4: and (3) purifying a product obtained after the reaction in the step 3:

uniformly mixing 0.8 x (50 mu L) AmpureXP magnetic beads with the amplification product, and placing the PCR tube on a magnetic frame for standing for 5min;

after the magnetic beads are completely adsorbed on the tube wall, removing the supernatant, washing the magnetic beads twice by using freshly prepared 80% ethanol, and removing the supernatant;

standing at room temperature for 3-5 min, and drying the magnetic beads (taking care not to dry the magnetic beads excessively so as not to influence the recovery efficiency);

adding 15 mu L of TE buffer, EB buffer or nuclease-free water to resuspend the magnetic beads according to the requirements of downstream experiments;

standing at room temperature for 3-5 min, placing the PCR tube on a magnetic frame, and sucking 12 mu L of supernatant, wherein the supernatant is double-stranded cDNA.

And 5: pre-amplification:

taking 12 mu L of the purified product to a PCR tube, and adding 30 mu L of the pre-amplification reaction mixed solution in the previous step to each sample;

the flash was then placed on ice and the samples were incubated on a PCR instrument under the conditions shown in table 4 below.

TABLE 4

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And 6: performing exponential amplification: adding 30 mu L of exponential amplification reaction mixed solution into the product of the previous reaction, simultaneously and respectively adding 1 mu L of tag sequence primers into each tube of reaction, uniformly mixing, centrifuging, and amplifying in a PCR instrument under the reaction conditions shown in Table 5.

TABLE 5

And 7: and (3) purification:

uniformly mixing 1 x (65 mu L) AmpureXP magnetic beads with the amplification product, and then placing the PCR tube on a magnetic frame for standing for 5min;

standing at room temperature for 3-5 min, and adding 25 μ L of TE buffer, EB buffer or nuclease-free water to resuspend the magnetic beads after the magnetic beads are dried (taking care not to excessively dry the magnetic beads so as not to influence the recovery efficiency) according to the requirements of downstream experiments;

standing at room temperature for 3-5 min, placing the PCR tube on a magnetic frame, and sucking 20 mu L of supernatant, wherein the supernatant is the sequencing library.

After the sequencing library is obtained, the quality detection of the library can be carried out. The method comprises the following specific steps:

mu.L of the purified amplification product was taken and 1. Mu.L of 6 XDNA sample buffer (KANG, century Biotech Co., ltd., product No. CW 0610A) was added to prepare a sample. 1% agarose gel was used, and GeneRuler 100bp Plus (Thermo Scientific) was used as a marker ^TM SM 0323). Referring to FIG. 7, the first lane from left to right is labeled with molecular weight, lanes 2-5 are 20, 10, 5, 3cells amplified products, and the products are concentrated around 300-500 bp.

Analysis of transcriptome and genomic copy number:

wherein, the analysis of the transcription group data is as follows:

the prepared library was sequenced on the computer, and the data size was 5M reads. The library was sequenced using a MGI 2000 sequencer, single-ended, reading 55bp in length. Transcriptome data results are shown in table 6, and table 6 is the main quality index of the transcriptome high throughput sequencing results.

TABLE 6

Therefore, the technical scheme provided by the application has the following advantages:

the initial amount of template is very low: 3cells can be used as an initial template for efficient amplification;

the amplification sensitivity is high: after a reaction system is optimized, more genes and transcripts can be detected after a lower reverse transcription-amplification cycle number;

and (3) reducing data preference: the complete transcriptome information can be obtained by combining polyT with polyA of mRNA and synthesizing cDNA, so that the preference of 3 'and 5' is avoided;

the success rate of the experiment is high: the cDNA is subjected to subsequent pre-amplification and exponential amplification to form a second-generation sequencing library, so that the treatment of a sample and the loss caused by the treatment are greatly reduced, and the success rate of an experiment is improved.

Wherein, the genome data are shown in table 7, and table 7 is the main quality index of the high-throughput sequencing result of the genome.

TABLE 7

The data in Table 7 show that 20cells, 10cells, 5cells, and 3cells can all have acceptable chromosome copy number measurements. As shown in fig. 8, each of chromosomes 1 to 22 in each experimental group had approximately two copies except for individual data points, and the sex chromosome X had approximately 2 copies and the Y chromosome had approximately 0 copy, which was consistent with the case of women with normal chromosome copy numbers.

In accordance with the above method embodiments, the present application also provides an electronic device, and an electronic device described below and a gene library constructing method described above are referred to in correspondence.

Referring to fig. 9, the electronic device includes:

a memory 332 for storing computer programs;

a processor 322 for implementing the steps of the gene library construction method of the above method embodiments when executing the computer program.

Specifically, referring to fig. 10, fig. 10 is a schematic diagram of a specific structure of an electronic device provided in this embodiment, which may generate relatively large differences due to different configurations or performances, and may include one or more processors (CPUs) 322 (e.g., one or more processors) and a memory 332, where the memory 332 stores one or more computer applications 342 or data 344. Memory 332 may be, among other things, transient or persistent storage. The program stored in memory 332 may include one or more modules (not shown), each of which may include a sequence of instructions operating on a data processing device. Still further, the central processor 322 may be configured to communicate with the memory 332 to execute a series of instruction operations in the memory 332 on the electronic device 301.

The electronic device 301 may also include one or more power sources 326, one or more wired or wireless network interfaces 350, one or more input-output interfaces 358, and/or one or more operating systems 341.

The steps in the gene library constructing method described above may be realized by the structure of an electronic device.

In accordance with the above method embodiments, the present application also provides a readable storage medium, and a readable storage medium described below and a gene library construction method described above are referred to in correspondence.

A readable storage medium, on which a computer program is stored, which, when executed by a processor, implements the steps of the gene library construction method of the above-described method embodiment.

The readable storage medium may be a usb disk, a removable hard disk, a Read-Only Memory (ROM), a Random Access Memory (RAM), a magnetic disk or an optical disk, and various other readable storage media capable of storing program codes.

The embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same or similar parts among the embodiments are referred to each other. As for the library building kit disclosed in the embodiment, the library building kit corresponds to the method disclosed in the embodiment, so that the description is simple, and the relevant points can be referred to the description of the method part.

Those of skill would further appreciate that the various illustrative components and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both, and that the various illustrative components and steps have been described above generally in terms of their functionality in order to clearly illustrate this interchangeability of hardware and software. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the technical solution. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present application.

The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. A software module may reside in Random Access Memory (RAM), memory, read-only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art.

Finally, it should be further noted that, in this document, relationships such as first and second, etc., are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Also, the terms include, or any other variation is intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

The principle and the implementation of the present application are explained herein by applying specific examples, and the above description of the embodiments is only used to help understand the method and the core idea of the present application; meanwhile, for a person skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and application scope, and in summary, the content of the present specification should not be construed as a limitation to the present application.

Claims

1. A method of constructing a gene library, comprising:

cracking cells to be detected to obtain a cracking product; the cleavage product has gDNA and mRNA which are cleaved to release the cells to be detected;

2. The method of claim 1, wherein the step of synthesizing the cDNA corresponding to the mRNA by adding the cleavage product to a reverse transcription reaction mixture containing a reverse transcription primer comprises:

3. The method of claim 2, wherein the step of synthesizing the second strand of cDNA using the first strand of cDNA as a template by a template switching primer under the polymerization activity of reverse transcriptase comprises:

4. The method of claim 1, further comprising, after synthesizing the cDNA corresponding to the mRNA and before constructing the gene library of the test cell based on the cDNA and the gDNA:

5. The method for constructing a gene library according to claim 1, wherein constructing a gene library of the test cell based on the cDNA and the gDNA comprises:

6. The method of claim 5, further comprising, after constructing the gene library of the test cells:

sequencing the gene library.

7. The method of claim 5, wherein the simultaneous amplification of the cDNA and the gDNA comprises:

and sequentially carrying out linear pre-amplification reaction and exponential amplification reaction by using the second cDNA chain and the gDNA as templates.

8. The method of claim 7, wherein the linear pre-amplification reaction is performed and comprises:

performing linear pre-amplification reaction in a pre-amplification reaction mixed solution by using the cDNA second chain and the gDNA as templates; wherein the pre-amplification reaction mixture comprises: a pre-amplification primer, a nucleotide monomer mixture, a nucleic acid polymerase and a primer having a complementary sequence or the same sequence as part or all of the sequencing universal primer; wherein the pre-amplification primer comprises a universal sequence and a variable sequence from the 5 'end to the 3' end.

9. The method of constructing a gene library according to claim 7 or 8, wherein an exponential amplification reaction is performed, comprising:

adding the exponential amplification reaction mixed solution into a linear pre-amplification reaction product to perform exponential amplification reaction; wherein the exponential amplification reaction mixture comprises: an exponential amplification primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the exponential amplification primer comprises, from 5 'end to 3' end, a specific sequence and a universal sequence.

10. A banking kit, comprising: the kit comprises a kit body and a library building reagent stored in the kit body;

wherein the poly (T) of the reverse transcription primer can be specifically and complementarily paired and combined with a poly (A) tail on mRNA in the total RNA of the cell to be detected;

the 3' end of the template switching primer has G of LNA matching the specified number;

the steps of implementing the method for constructing a gene library according to any one of claims 1 to 9 based on the library construction kit.

11. The banking kit according to claim 10, wherein the banking reagent further comprises: at least one of a surfactant, a lyase, and an inhibitor that inhibits RNA degradation.

12. An electronic device, comprising:

a memory for storing a computer program;

a processor for implementing the steps of the method of constructing a gene library according to any one of claims 1 to 9 when executing the computer program.

13. A readable storage medium, wherein a computer program is stored thereon, and when executed by a processor, the computer program implements the steps of the method for constructing a gene library according to any one of claims 1 to 9.