Transcriptome library construction method based on multiple displacement amplification

Abstract

Provided are a transcriptome library construction method based on multiple displacement amplification, a kit used for said method, and a transcriptome sequencing method and a spatial transcriptome sequencing method based on this method. The method comprises: reverse-transcribing an mRNA molecule to obtain a cDNA chain; using the cDNA chain as a template, performing multiple displacement amplification under the actions of MDA primers and a DNA polymerase having chain displacement activity, to obtain multiple cDNA amplified fragments of different lengths, the cDNA amplified fragments constituting a transcriptome library, there being multiple MDA primers, and the MDA primers containing a first sequence used for sequencing and a second sequence used to bind to the cDNA chain, the second sequence being a random sequence, the second sequence randomly binding to the cDNA chain, and, optionally, the first sequence being a first linker, and the second sequence also containing degenerate bases.

Description

Transcriptome library construction method based on multiple displacement amplification Technical Field

[0001] The present application relates to the technical field of transcriptome sequencing, and in particular to a method for constructing a transcriptome library based on multiple displacement amplification. Background Art

[0002] Spatial transcriptome sequencing is a technology dedicated to quantifying the spatial mapping of transcriptional information. Building on traditional transcriptome sequencing, it provides detailed spatial information, helping researchers identify the location of transcripts within tissues at cellular levels or even higher resolution.

[0003] For spatial transcriptome sequencing, 10×Genomics, Wancheng, BGI, etc. have all launched related technologies. Among these related technologies, the construction of mRNA libraries is based on reverse transcription after mRNA capture to obtain cDNA, and the cDNA is sheared using physical methods (such as ultrasound, etc.) and enzymatic methods; some use in vitro transcription to re-transcribe cDNA to obtain amplified RNA (aRNA), and use metal ions to shear the aRNA to obtain a length suitable for sequencing. However, the processes required by these shearing methods are complex, have harsh experimental conditions, are expensive, and it is difficult to ensure the uniformity of the sheared products.

[0004] Therefore, there is an urgent need to provide a simple, easy and stable mRNA library construction solution.

[0005] Summary of the Invention

[0006] The embodiments of the present application provide a transcriptome library construction method based on multiple displacement amplification, a kit for use in the method, and a spatial transcriptome library construction method of a biological sample based on the method, a transcriptome sequencing method of a biological sample, and a spatial transcriptome sequencing method of a biological sample.

[0007] The first aspect of the present application provides a method for constructing a transcriptome library based on multiple displacement amplification, comprising: reverse transcribing mRNA molecules to obtain cDNA chains; using the cDNA chains as templates, performing multiple displacement amplification under the action of MDA primers and a DNA polymerase with chain displacement activity to obtain multiple cDNA amplified fragments of different lengths, wherein the cDNA amplified fragments constitute the transcriptome library; wherein there are multiple MDA primers, and the MDA primers comprise a first sequence for sequencing and a second sequence for binding to the cDNA chains, the second sequence being a random sequence, and the second sequence randomly binds to the cDNA chains.

[0008] In some embodiments, the first sequence is a first linker, and the second sequence further comprises degenerate bases.

[0009] In an embodiment of the present application, the reverse transcription of the mRNA molecule to obtain the cDNA chain includes: using the mRNA molecule as a template, performing reverse transcription under the action of a reverse transcription primer and a reverse transcriptase to obtain the cDNA chain, wherein the reverse transcription primer comprises a third sequence for capturing the mRNA and an optional fourth sequence for labeling the mRNA.

[0010] In some embodiments, the third sequence is oligothymidine (Oligo dT), and the fourth sequence is a unique molecular identifier sequence (UMI).

[0011] In an embodiment of the present application, the reverse transcription primer further comprises a fifth sequence for sequencing. In some embodiments, the fifth sequence is a second linker.

[0012] In the embodiment of the present application, after obtaining multiple cDNA amplified fragments of different lengths, the method further includes: performing secondary amplification on the multiple cDNA amplified fragments of different lengths based on the MDA primer or its fragment and the optional reverse transcription primer or its fragment.

[0013] In the embodiment of the present application, after obtaining a plurality of amplified cDNA fragments of different lengths, the method further comprises: screening the plurality of amplified cDNA fragments of different lengths to obtain the transcriptome library.

[0014] In the examples of the present application, the screening is performed using a solid phase carrier to obtain cDNA amplified fragments of a desired length as the transcriptome library. In some embodiments, the solid phase carrier is a silica-based magnetic bead. In some embodiments, the desired length is 200-800 nt, preferably 250-700 nt, and more preferably 250-600 nt.

[0015] In an embodiment of the present application, after the reverse transcription and before the multiple displacement amplification, the method further comprises: enriching the cDNA chain through a template switching mechanism.

[0016] In some embodiments, the cDNA chain is enriched by a template switching mechanism, specifically comprising: adding a sixth sequence for template switching to the 3' end of the cDNA chain; obtaining an extended chain of the cDNA chain by template switching based on a template switching oligonucleotide and the cDNA chain with the sixth sequence added to the end, wherein the template switching oligonucleotide comprises a seventh sequence for binding to the sixth sequence; and amplifying the extended chain to enrich the cDNA chain.

[0017] In some embodiments, the sixth sequence is 1-20 nt in length, preferably 2-10 nt in length, and in some embodiments, the sixth sequence is polycytosine. In some embodiments, the seventh sequence is polyguanine.

[0018] In an embodiment of the present application, the template switching oligonucleotide further comprises an eighth sequence, wherein the extended chain is amplified based on the eighth sequence to enrich the cDNA chain.

[0019] In the embodiments of the present application, the length of the first sequence in the MDA primer is 10-35 nt, preferably 10-25 nt. In some embodiments, the length of the second sequence is 4-15 nt, preferably 6-10 nt, and the degenerate base ratio in the second sequence is 100% or less, preferably greater than 75%, and more preferably greater than 80%.

[0020] In an embodiment of the present application, the method further comprises: preparing DNA nanoballs by rolling circle amplification based on the transcriptome library.

[0021] In the embodiments of the present application, the DNA polymerase having strand displacement activity is selected from one or more of the following: Klenow DNA polymerase, reverse transcriptase, Phi29 DNA polymerase, Bst DNA polymerase and Bsu DNA polymerase.

[0022] The second aspect of the present application provides a kit for constructing a transcriptome library based on multiple displacement amplification, comprising: an MDA reagent and a reverse transcription reagent; and optional fragment screening reagents and DNA nanoball preparation reagents, wherein the MDA reagent comprises an MDA primer and a DNA polymerase with chain displacement activity, the MDA primer comprises a first sequence for sequencing and a second sequence for binding to a cDNA chain, the second sequence is a random sequence, and the second sequence randomly binds to the cDNA chain.

[0023] In some embodiments, the first sequence is a first linker, and the second sequence further comprises degenerate bases.

[0024] In the embodiments of the present application, the length of the first sequence in the MDA primer is 10-35 nt, preferably 10-25 nt. In some embodiments, the length of the second sequence is 4-20 nt, preferably 6-10 nt, and the degenerate base percentage in the second sequence is 100% or less, preferably greater than 75%, and more preferably greater than 80%. In some embodiments, the MDA primer is selected from one or two of SEQ ID NOs: 1-2.

[0025] In the embodiments of the present application, the DNA polymerase having strand displacement activity is selected from one or more of the following: Klenow DNA polymerase, reverse transcriptase, Phi29 DNA polymerase, Bst DNA polymerase and Bsu DNA polymerase.

[0026] In the embodiment of the present application, the reverse transcription reagent comprises a reverse transcription primer, and the reverse transcription primer comprises a third sequence for capturing mRNA and an optional fourth sequence for labeling mRNA.

[0027] In some embodiments, the third sequence is oligothymidine (Oligo dT), and the fourth sequence is a unique molecular identifier (UMI). In some embodiments, the reverse transcription primer further comprises a fifth sequence for sequencing, and in some embodiments, the fifth sequence is a second adapter.

[0028] In the examples of the present application, the fragment screening reagent comprises a solid phase support for screening the cDNA amplified fragments to obtain cDNA amplified fragments of a desired length as the transcriptome library. In some embodiments, the solid phase support is a silica-based magnetic bead. In some embodiments, the desired length is 200-800 nt, preferably 250-700 nt, and more preferably 250-600 nt.

[0029] In an embodiment of the present application, the kit further comprises a template switching reagent, wherein the template switching reagent comprises a template switching oligonucleotide, and the template switching oligonucleotide comprises a seventh sequence for binding to the 3' end of the cDNA chain. In some embodiments, the seventh sequence is GGG.

[0030] In some embodiments, the template-switching oligonucleotide further comprises an eighth sequence for amplifying the extended strand.

[0031] The third aspect of the present application provides a method for constructing a spatial transcriptome library of a biological sample, comprising: (1) capturing mRNA molecules from the biological sample in situ; and (2) constructing a spatial transcriptome library according to the transcriptome library construction method based on multiple displacement amplification as described in any embodiment of the first aspect of the present application, wherein a spatial coordinate sequence for locating the spatial position of the mRNA molecule in the biological sample is introduced into the cDNA chain to obtain a spatial transcriptome library.

[0032] In some embodiments, the spatial coordinate sequence is introduced into the cDNA chain by making the reverse transcription primer contain the spatial coordinate sequence.

[0033] In an embodiment of the present application, in situ capturing of mRNA molecules from the biological sample specifically includes: placing the biological sample on the surface of a capture chip; fixing and permeabilizing the biological sample to release the mRNA molecules; and using the capture chip to capture the mRNA molecules in situ, wherein the capture chip is connected to the reverse transcription primer.

[0034] The fourth aspect embodiment of the present application proposes a transcriptome sequencing method for a biological sample, comprising: constructing a transcriptome library according to the transcriptome library construction method based on multiple displacement amplification as described in any embodiment of the first aspect of the present application; and sequencing the transcriptome library to obtain transcriptome data of the biological sample.

[0035] The fifth aspect embodiment of the present application proposes a spatial transcriptome sequencing method for a biological sample, comprising: constructing a spatial transcriptome library according to the spatial transcriptome library construction method based on multiple displacement amplification as described in any embodiment of the third aspect of the present application; and sequencing the spatial transcriptome library to obtain spatial transcriptome data of the biological sample, wherein the spatial position information of the mRNA molecule in the biological sample is obtained according to the spatial coordinate sequence.

[0036] In the embodiment of the present application, the biological sample is a tissue section or a single cell sample.

[0037] The technical solution of this application achieves the following technical effects:

[0038] The transcriptome library construction method based on multiple displacement amplification in the embodiment of the present application can realize the preliminary fragmentation of long-chain cDNA by designing MDA primers and based on multiple displacement amplification. This method can effectively replace the library construction method or in vitro transcription library construction method comprising traditional physical interruption (such as using ultrasound, etc.), transposase (such as Tn5 enzyme) interruption, avoiding the introduction of ultrasound, enzyme, etc. in the traditional method, resulting in uneven fragment size, unstable library construction efficiency and other problems, and also avoiding the shortcomings such as unstable single-stranded RNA, high replication error rate and subsequent connection efficiency with ssDNA in the in vitro transcription method library construction. The transcriptome library construction method in the embodiment of the present application and the sequencing method based thereon have simple experimental procedures, few interfering factors, and stable results, and can obtain experimental results comparable to or even better than traditional methods, and have good adaptability to different types of tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0040] FIG1 is a method for constructing a transcriptome library based on multiple displacement amplification according to an embodiment of the present application;

[0041] FIG2 is another method for constructing a transcriptome library based on multiple displacement amplification according to an embodiment of the present application;

[0042] FIG3 shows the visualization results of mRNA captured according to Example 3 of the present application;

[0043] FIG4 is a comparison of the number and type of genes sequenced from the control group library and the experimental group library according to Example 3 of the present application;

[0044] FIG5 shows the correlation between genes sequenced from the control group library and the experimental group library according to Example 3 of the present application. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0046] This application is made based on the following knowledge of the inventors:

[0047] Spatial omics (including spatial transcriptomics, spatial proteomics, and spatial immunoomics) enables the interpretation of cell fate determination in both temporal and spatial dimensions. These multi-omics technologies involve the use of targeted binding to known target multi-omics substances, followed by imaging detection, to obtain spatially localized multi-omics information, or by in situ capturing of corresponding multi-omics information with random probes with spatial coordinates, followed by conversion to cDNA for next-generation sequencing to simultaneously obtain the spatial location coordinates of the captured products.

[0048] Several sequencing companies have launched spatial transcriptome analysis technologies, such as BGI's Stereo-seq technology, which uses its own DNBSEQ technology to load probes with spatial position information onto biochips with nanometer resolution through sequencing. These biochips capture target nucleic acids or proteins in biological tissues, and then convert the captured information into corresponding nucleotide sequences through biochemical reactions and recover them. Finally, the captured information and spatial coordinates are matched one by one through second-generation sequencing (Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays[J].Cell,2022,185(10):1777-1792.e21.DOI:10.1016 / j.cell.2022.04.003.).

[0049] However, there are many deficiencies in the transcriptome library construction scheme in the related art, especially in the step of shearing cDNA or aRNA. Specifically, the amplification of the cDNA template chain is mainly carried out by the template switching method, or the Poly A labeling method, and the amplified template is sheared by physical or enzymatic methods to obtain a library of suitable length for sequencing; and if the in vitro transcription method is adopted, the obtained aRNA is sheared by metal ions to obtain a length suitable for sequencing. However, the process of using the physical shearing method to build a library is complicated, and the product needs to be end-filled after shearing (i.e., the 3' end and the 5' end are tailed and modified respectively) to facilitate the addition of sequencing adapters, and the reaction efficiency of each link is low. For enzymatic shearing library construction, the Tn5 transposase shearing library construction method is currently commonly used. The disadvantages of Tn5 fragmentation library construction are that, before fragmentation, Tn5 must first be primer-embedded, and the embedding effect of this step will affect the subsequent fragmentation efficiency. In addition, the Tn5 enzyme is an enzyme dosage-dependent fragmentation enzyme and will not detach from the DNA chain until the reaction system is terminated. Therefore, it has high requirements for DNA quantification. If the ratio of the two is not appropriate, it is difficult to achieve a good fragmentation effect. Furthermore, impurities in the solution can affect the insertion of the Tn5 enzyme, and some DNA-binding proteins can also affect the uniform insertion of the adapter sequence. In addition, because the insertion site of Tn5 has certain sequence preferences, it may affect the uniformity of the final fragmentation, resulting in poor stability of the final reaction. In vitro transcription library construction requires the use of RNA polymerase, and the resulting product is RNA, which is not stable and has a high replication error rate. In addition, linear ligation of ssDNA and RNA is required, which is inefficient and complex.

[0050] Based on this, the inventors have developed a simple, easy-to-use and stable mRNA library construction scheme after multiple experiments and tests. This scheme can replace traditional long-chain fragmentation methods such as transposase and mechanical fragmentation. By using random primers or non-completely random primers (i.e., their 5' ends are fixed sequences) to perform multi-site hybridization with cDNA templates, and using a DNA polymerase with chain displacement function to perform polymerization reactions, single-stranded cDNA fragments of different lengths based on the same template are obtained. The target fragments are then enriched by PCR; after that, solid phase carriers such as magnetic beads or gels are used to screen the fragments to obtain DNA fragments of a length suitable for sequencing. The mRNA library construction scheme proposed in the embodiment of the present application is based on random primer hybridization, DNA chain displacement polymerase, and multiple displacement amplification to obtain cDNA single chains of different lengths. No physical shearing or introduction of shearing enzyme is required, avoiding complex processes such as end repair after physical shearing, as well as the pre-linker embedding treatment when using shearing enzyme and the sequence preference of shearing enzyme for the selection of shearing sites during work, making the process simpler and the resulting product more uniform; at the same time, the scheme of the present application can replace the in vitro transcription scheme, during which the use of RNA polymerase is avoided, and the obtained product is a DNA library (which can be ssDNA), so the product stability of each stage is better; in addition, the MDA system of the embodiment of the present application can realize one-step library construction by directly introducing sequencing primers using designed MDA primers or reverse transcription primers, and the fragment length screening by magnetic beads or the like in the later stage can obtain the machine library, which has the advantages of simple process, few interference factors, and more stable sequencing results.

[0051] The transcriptome library construction method based on multiple displacement amplification (MDA) proposed in the embodiments of the present application may include the following steps: S1-S4.

[0052] S1: Reverse transcription of mRNA molecules to obtain cDNA chains.

[0053] In the embodiments of the present application, reverse transcription can be performed based on a reverse transcription primer using an mRNA molecule as a template to obtain a cDNA chain, wherein the reverse transcription primer can include a capture sequence (i.e., a third sequence) for capturing the mRNA. In some embodiments, the capture sequence can be an oligothymidine (Oligo dT) that is complementary to the poly A tail of the mRNA. The reverse transcription primer can also include an identifier (i.e., a fourth sequence) for labeling the mRNA, which can be a unique molecular identifier (UMI) sequence. It is understood that by introducing a UMI into each cDNA chain through reverse transcription, products amplified from the same cDNA in the subsequent library construction process all carry the same label, while naturally occurring repeat fragments carry different labels. Later, by filtering the data using the UMI, the cDNA in the sample can be accurately counted, thereby distinguishing between sequence multiple copies and false multiple copies (caused by PCR bias or other factors).

[0054] In certain embodiments, the reverse transcription primer may further comprise a sequence (i.e., a fifth sequence) for binding to a sequencing primer. In certain embodiments, the fifth sequence may be a joint. It is understood that, based on such a reverse transcription primer structure, a joint may be introduced directly to one end of the cDNA during reverse transcription, thereby greatly simplifying the library construction process.

[0055] S2: Using the cDNA chain as a template, multiple displacement amplification is performed under the action of MDA primers and a DNA polymerase with chain displacement activity to obtain multiple cDNA amplified fragments of different lengths, which constitute the transcriptome library.

[0056] In the embodiments of the present application, preliminary fragmentation of long-chain cDNA can be achieved by designing MDA primers and based on multiple displacement amplification. In some embodiments, there are multiple MDA primers, and the MDA primers include a sequence for sequencing that can bind to a subsequent sequencing primer (i.e., a first sequence) and a second sequence for binding to the cDNA chain, wherein the first sequence is located at the 5' end of the MDA primer and the second sequence is located at the 3' end of the MDA primer. In some embodiments, the length of the first sequence in the MDA primer is 10-35 nt, preferably 10-25 nt.

[0057] In some embodiments, the first sequence can be a linker. It is understood that based on such an MDA primer structure, a linker can be introduced directly at the other end of the generated cDNA amplified fragment in multiple displacement amplification, thereby greatly simplifying the library construction process. It should be noted that the linker in the embodiments of the present application can be a linker designed as needed or a commercially available linker, as long as it can be used for subsequent sequencing. This application is not intended to limit the sequence, length, or modifications of the linker.

[0058] In some embodiments, the second sequence can be a completely random sequence or a partially random sequence, designed to bind to multiple positions of the cDNA chain. A completely random sequence means that all bases in the second sequence are randomly generated; a partially random sequence means that the base types at some positions in the second sequence can be pre-set, while the base types at the remaining positions are randomly generated. In some embodiments, the second sequence can also include degenerate bases, for example, R, Y, M, K, S, W, H, B, V, D, N, or any combination thereof. In some embodiments, the length of the second sequence can be 4-15 nt, preferably 6-10 nt, and more preferably 6-8 nt. In some embodiments, the degenerate base percentage of the second sequence is 100%, i.e., all bases in the second sequence are degenerate bases. In other embodiments, the degenerate base percentage of the second sequence can be less than 100%, i.e., some bases in the second sequence are degenerate bases, and the remaining base types are selected from any of A / T / G / C. In some embodiments, the degenerate base percentage of the second sequence is less than 100%, preferably greater than 75%, and more preferably greater than 80%. It is understood that, based on the random structure of the second sequence, the MDA primers can randomly bind to multiple positions on the cDNA chain to amplify multiple cDNA fragments of varying lengths. These cDNA fragments also contain sequencing adapters (i.e., the first sequence), thereby achieving fragmentation and adapter addition in a single step. The resulting cDNA fragments of varying lengths form a preliminary library ready for sequencing, or can be sequenced after simple size screening.

[0059] In some embodiments, the second sequences of the multiple MDA primers are identical to each other. In other embodiments, the second sequences of the multiple MDA primers are completely different or partially different from each other, so as to better bind to multiple different positions on the cDNA chain.

[0060] In some specific embodiments, the MDA primers are selected from one or a combination of two of SEQ ID NOs: 1-2. It is understood that by designing highly efficient and stably binding MDA primers (e.g., SEQ ID NOs: 1-2 shown in Table 1, where the underlined portion is a random sequence portion), the efficiency of multiple displacement amplification is effectively improved, achieving efficient production of multiple cDNA amplified fragments of different lengths.

[0061] Table 1 MDA primer sequences

[0062] The method of multiple displacement amplification using MDA primers proposed in the examples of this application can effectively replace traditional physical fragmentation (such as using ultrasound, etc.) and transposase (such as Tn5 enzyme) fragmentation, and solves many problems in traditional fragmentation methods, such as complex experimental operations, uneven fragment lengths, and unstable sequencing results.

[0063] In the examples of the present application, a DNA polymerase with a chain displacement function is used to perform multiple displacement amplification. In some embodiments, the DNA polymerase can be selected from one or more of the following: Klenow DNA polymerase, reverse transcriptase, Phi29 DNA polymerase, Bst DNA polymerase, and Bsu DNA polymerase. It is understandable that the DNA polymerase in the examples of the present application does not need to be pretreated in advance and can be directly used in the preparation of the MDA system; at the same time, the reaction system and reaction conditions of the DNA polymerase are simple, for example, it is only necessary to control the isothermal amplification, thereby simplifying the experimental operation while ensuring the amplification efficiency. It should be noted that the DNA polymerase used in the examples of the present application can also be other polymerases with a chain displacement function, as long as it can catalyze the completion of multiple displacement amplification, and the present application has no intention of limiting its type, conditions of use, and system preparation.

[0064] In the embodiment of the present application, after obtaining a plurality of amplified cDNA fragments of different lengths, the method further comprises: screening the plurality of amplified cDNA fragments of different lengths to obtain a transcriptome library.

[0065] In the examples of the present application, the cDNA amplified fragments of different lengths obtained after multiple displacement amplification based on MDA primers are transcriptome libraries with sequencing conditions connected to sequencing adapters. They can be subsequently screened for length, and the screened cDNA amplified fragments with the desired length are used as transcriptome libraries, where the desired length refers to the length that meets the requirements of the sequencing platform and is suitable for sequencing on the machine. In some embodiments, the desired length can be 200-800nt, preferably 250-700nt, and more preferably 250-600nt.

[0066] In certain embodiments, solid phase carrier is used to screen fragment length, wherein solid phase carrier can be solid magnetic beads, chip or gel etc. with DNA affinity group, such as silica-based magnetic beads, agarose gel etc. It is understandable that, by controlling the ratio of the affinity group of solid phase carrier and the amount of cDNA fragment to be screened, solid phase carrier specific adsorption can be made to have the cDNA amplified fragment of desired length, realize the screening of fragment length with this. In certain embodiments, the screening of sequencing library can be realized by magnetic bead double selection. The transcriptome library construction method proposed in the present application embodiment uses solid phase carrier to screen fragment length, and this method has the advantages such as simple process, few interfering factors and stable result.

[0067] In the present application embodiment, can carry out subsequent sequencing to transcriptome library based on the joint sequence in MDA primer and / or reverse transcription primer or its fragment, wherein sequencing can be single-end sequencing or double-end sequencing.In certain embodiments, after step S2, before step S3, the transcriptome library construction method based on multiple displacement amplification (MDA) can also include: based on MDA primer or its fragment and optional reverse transcription primer or its fragment, the cDNA amplified fragment different in multiple lengths is carried out secondary amplification.It is understandable that this secondary amplification can be carried out two-strand amplification for template with the single-stranded cDNA of multiple lengths that MDA generates, to obtain the double-stranded DNA library different in multiple lengths.Equally, can use the joint sequence in MDA primer and / or reverse transcription primer or its fragment as a chain sequencing primer and / or two-chain sequencing primers to carry out single-end or double-end sequencing to this double-stranded DNA library. The transcriptome library construction method of the embodiment of the present application obtains a more stable double-stranded DNA library by using the 5' terminal sequence (such as the first sequence) of the MDA primer introduced in the MDA step as a one-side primer and coordinating it with a fixed sequence (such as the fifth sequence) in the reverse transcription primer as the other-side primer; at the same time, the partial sequence (such as the first sequence) of the MDA primer introduced in the MDA step and the partial sequence (such as the fifth sequence) of the reverse transcription primer introduced in the reverse transcription step can be used as a second-strand sequencing primer and a single-strand sequencing primer, respectively, thereby simplifying the experimental steps and improving the efficiency of library construction.

[0068] In an embodiment of the present application, after step S1 and before step S2, the transcriptome library construction method based on multiple displacement amplification further includes: performing template conversion based on the cDNA chain, and performing subsequent multiple displacement amplification using the cDNA chain obtained after the conversion as a template; that is, enriching the cDNA chain by a template conversion mechanism. To this end, a DNA polymerase having terminal transferase activity (e.g., Moloney murine leukemia virus (MMLV) reverse transcriptase) can be added to the reverse transcription system during reverse transcription to add a sixth sequence for template conversion to the 3' end of the cDNA chain. In some embodiments, the length of the sixth sequence can be 1-20nt, preferably 2-10nt. In some embodiments, the sixth sequence is polycytosine (Poly C), such as CCC. It is understood that, based on a cDNA chain with a sixth sequence added to its end, template switching can be performed using a template switching oligonucleotide (TSO) under the action of a reverse transcriptase (e.g., MMLV reverse transcriptase), wherein the TSO comprises a seventh sequence for binding to the sixth sequence and an eighth sequence for subsequent amplification. When the sixth and seventh sequences are complementary to each other, the MMLV reverse transcriptase will continue reverse transcription using the TSO as a template, thereby obtaining an extended cDNA chain. Subsequently, based on the eighth sequence in the extended chain (e.g., using a primer complementary to the eighth sequence), the extended chain is amplified to enrich the cDNA chain for subsequent multiple displacement amplification. In some embodiments, the seventh sequence can be polyguanosine (Poly G), such as rGrGrG. In other embodiments, other terminal transferases can be used for the template switching reaction, and the added sixth sequence can be polyadenine (Poly A).

[0069] It is understandable that the transcription library construction method in the embodiment of the present application is by template switching, can obtain the complete 5 ' end sequence comprising transcript, and can carry out efficient enrichment to cDNA before multiple displacement amplification, thus effectively improving the construction effect of cDNA library.In the embodiment of the present application, after obtaining transcriptome library, DNA nanometer ball (DNB) can also be prepared by rolling circle amplification, and this DNB is sequenced to obtain transcriptome data.It is understandable that by further preparing double-stranded transcriptome library as DNB, can effectively enhance sequencing signal intensity and improve sequencing accuracy, realize high-precision high-quality sequencing.

[0070] The transcriptome library construction method based on multiple displacement amplification proposed in the present application embodiment can achieve the preliminary fragmentation of long-chain cDNA by designing MDA primers and based on multiple displacement amplification. This method can effectively replace the library construction method or in vitro transcription library construction method comprising traditional physical interruption (such as using ultrasound, etc.) and transposase (such as Tn5 enzyme) interruption, avoiding the problems such as uneven fragment size and unstable library construction efficiency caused by the introduction of ultrasound, enzyme, etc. in the traditional method, and also avoiding the shortcomings such as unstable single-stranded RNA, high replication error rate and subsequent connection efficiency with ssDNA in the in vitro transcription method library construction. The transcriptome library construction method in the present application embodiment has a simple experimental process, few interfering factors, and stable results, and can obtain experimental results comparable to or even better than traditional methods, and has good adaptability to different types of tissues.

[0071] Figure 1 illustrates a transcriptome library construction method based on multiple displacement amplification according to an embodiment of the present application. This method can be used to construct a library for mRNA captured by a PolyT probe with a UMI. As shown in Figure 1, the specific library construction process is as follows: a. Reverse transcription: mRNA with a poly dA tail is reverse transcribed using an mRNA hybridization probe (also known as a reverse transcription primer), where the mRNA hybridization probe contains a poly T sequence for capturing mRNA (corresponding to the third sequence), a UMI for labeling mRNA molecules (corresponding to the fourth sequence), and a fixed sequence for subsequent amplification and sequencing (i.e., Library F Primer, corresponding to the fifth sequence). Accordingly, the cDNA chain obtained in step a sequentially contains a cDNA sequence complementary to the mRNA, a PolyT sequence, a UMI, and a Library F Primer (which can also serve as a linker). b.MDA: Using the obtained cDNA chain as a template, an MDA primer containing a specific 5' end sequence (corresponding to the first sequence) is used to hybridize with the template, wherein the completely random or partially random sequence contained in the MDA primer (corresponding to the second sequence, represented by NNN in the figure) randomly binds to the cDNA chain, and a polymerization reaction catalyzed by a DNA polymerase with a chain displacement function is obtained to obtain multiple single-stranded cDNA copies of different lengths. The 5' and 3' ends of these single-stranded cDNA copies of different lengths have known sequences that can be used for subsequent amplification or sequencing (i.e., Library F Primer and the first sequence of the MDA primer, which may correspond to Library R Primer in the figure), and contain sequences corresponding to the transcripts at the 3' end of the original mRNA. Steps a and b achieve fragmentation and adapter ligation in the construction of the transcriptome library, and these single-stranded cDNA copies of different lengths containing sequencing fragment elements are actually in the form of preliminary libraries. c. Library PCR: Using the MDA primer 5' end sequence (i.e., Library R Primer) in combination with Library F Primer, these single-stranded cDNA copies of different lengths are amplified to obtain a double-stranded DNA library corresponding to the mRNA 3' end sequence.

[0072] In some embodiments, the method may further include d. library screening: performing magnetic bead double selection on the obtained double-stranded DNA library to obtain a library with fragment lengths suitable for sequencing. The library is then sequenced and analyzed to obtain the mRNA sequence.

[0073] The method of multiple displacement amplification using MDA primers proposed in the examples of this application can effectively replace traditional physical fragmentation (such as using ultrasound, etc.) and transposase (such as Tn5 enzyme) fragmentation, and solves many problems of traditional fragmentation methods, such as complex experimental operations, uneven fragment lengths, and unstable sequencing results. At the same time, through primer design, sequencing adapters can be directly introduced into the amplified fragments while achieving fragmentation, thereby greatly simplifying the library construction process.

[0074] Figure 2 is another transcriptome library construction method based on multiple displacement amplification according to an embodiment of the present application, which can be applied to transcriptome sequencing or spatial transcriptome sequencing in related technologies, such as a capture chip fixed with an mRNA capture probe or containing a spatial coordinate (CID) sequence (e.g., BGI STOmics transcriptome chip of Huada). As shown in Figure 2, the transcriptome library construction method for the capture chip, based on the steps ad described in the above embodiment, further includes: a'. Template conversion: by adding MMLV-RT enzyme to the system, after reverse transcription to obtain a cDNA chain, using MMLV-RT enzyme to add a Poly C sequence (corresponding to the sixth sequence, CCC sequence in the figure) to the 3' end of the cDNA chain, and then based on the cDNA chain with the Poly C sequence at the end, template conversion and cDNA chain extension are performed under the guidance of a template switch primer (Template Switch Oligo, TSO), wherein TSO comprises a Poly G sequence (corresponding to the seventh sequence) that can bind to the Poly C sequence and a primer binding region TSO' sequence (corresponding to the eighth sequence) for subsequent PCR amplification. Subsequently, MDA is performed on the cDNA strand obtained after template switching (i.e., steps bd). In some embodiments, after the template switching step, the method may further include step a', using the extended cDNA strand obtained in step a' as a template and adding primers that complementarily bind to both ends of the extended cDNA strand for PCR to enrich the target transcriptome and remove interference from impurity sequences in the system. The remaining steps of the transcriptome library construction method based on multiple displacement amplification shown in Figure 2, including reverse transcription, MDA, library PCR, and fragment screening, are the same as steps ad described above and are not further described here.

[0075] Therefore, the transcriptome library construction method based on multiple displacement amplification according to the embodiment of the present application can also be combined with technologies such as capture chips in current transcriptome sequencing, and can be widely used in transcriptome sequencing platforms; at the same time, by introducing a template conversion step, the complete 5' end information of mRNA can be effectively obtained, thereby achieving higher-precision transcriptome detection.

[0076] The embodiments of the present application also propose a method for constructing a spatial transcriptome library of a biological sample, comprising: (1) capturing mRNA molecules from the biological sample in situ; (2) constructing a spatial transcriptome library according to the transcriptome library construction method based on multiple displacement amplification described in any of the above embodiments, wherein a spatial coordinate (CID) sequence for locating the spatial position of the mRNA molecule in the biological sample is introduced into the cDNA chain to obtain the spatial transcriptome library.

[0077] As shown in Figure 2, the transcriptome library construction method based on multiple displacement amplification proposed in the embodiments of the present application can also be used for library construction and sequencing of spatial transcriptomes. Specifically, based on steps a, a', a", and bd described in the above embodiments, the method further includes: introducing a CID sequence into the cDNA chain to indicate the spatial position of the mRNA molecule in the biological sample. In some embodiments, the CID sequence can be pre-linked to an mRNA capture probe (which can correspond to a reverse transcription primer) so that the CID sequence can be introduced into the cDNA chain by reverse transcription after the mRNA is captured in situ. It can be understood that by sequencing the transcriptome library constructed by in situ capture of mRNA and loading the CID sequence and analyzing its transcriptome data, the spatial position information of the mRNA molecule can be obtained based on the CID sequence, thereby restoring the in situ expression of the mRNA molecule at the level of each tissue or cell in the biological sample. The remaining steps of the spatial transcriptome library construction method based on multiple displacement amplification proposed in the embodiments of the present application, including reverse transcription, MDA, library PCR and fragment screening, are the same as steps ad described above, and can also be combined with steps a', a", etc., and will not be repeated here.

[0078] In an embodiment of the present application, the in situ capture of mRNA molecules may include: placing a biological sample on the surface of a capture chip; fixing and permeabilizing the biological sample to release the mRNA molecules; and using a capture chip to capture the mRNA molecules in situ, wherein the capture chip is connected to a reverse transcription primer. In some embodiments, the biological sample can be a biological tissue section or a single cell sample, which can be fresh or frozen. It should be noted that the capture chip in the embodiment of the present application and the sample fixation, permeabilization and other operations based on the chip can achieve in situ capture of mRNA, and the present application is not intended to be limiting.

[0079] The method for constructing a spatial transcriptome library of a biological sample proposed in the embodiment of the present application further introduces a spatial coordinate sequence into the cDNA chain on the basis of traditional transcriptome library construction, effectively realizing the restoration and analysis of the spatial position of mRNA; at the same time, this method is also based on MDA amplification of multiple cDNA fragments of different lengths to replace traditional physical shearing (such as using ultrasound, etc.) and transposase (such as Tn5 enzyme) shearing, effectively solving many problems in traditional shearing methods, such as complex experimental operations, uneven sheared fragment lengths, and unstable sequencing results.

[0080] The embodiments of the present application also provide a method for transcriptome sequencing of a biological sample, comprising: constructing a transcriptome library according to the transcriptome library construction method based on multiple displacement amplification described in any of the above embodiments; and sequencing the transcriptome library to obtain transcriptome data of the biological sample.

[0081] An embodiment of the present application also proposes a spatial transcriptome sequencing method for a biological sample, comprising: constructing a spatial transcriptome library according to the spatial transcriptome library construction method based on multiple displacement amplification described in any of the above embodiments; and sequencing the spatial transcriptome library to obtain spatial transcriptome data of the biological sample, wherein the spatial position information of the mRNA molecule in the biological sample is obtained according to the CID sequence.

[0082] In some embodiments, the sequencing of the transcriptome library or the spatial transcriptome library can be single-end sequencing or double-end sequencing, wherein the first sequence and / or the fifth sequence can be used to combine with a sequencing primer. In some embodiments, sequencing is performed using a sequencer based on DNB sequencing technology.

[0083] In response to the transcriptome library construction method based on multiple displacement amplification and the spatial transcriptome library construction method proposed in the above embodiments, the embodiments of the present application also propose a kit for constructing a transcriptome library based on multiple displacement amplification, comprising: an MDA reagent and a reverse transcription reagent, as well as an optional fragment screening reagent and an optional DNB preparation reagent, wherein the MDA reagent includes an MDA primer and a DNA polymerase with chain displacement activity, the MDA primer comprises a first sequence for sequencing and a second sequence for binding to a cDNA chain, the second sequence is a random sequence, and the second sequence randomly binds to the cDNA chain. In some embodiments, the first sequence is a first linker, and the second sequence also comprises a degenerate base.

[0084] It should be noted that the explanation of the embodiment of the transcriptome library construction method based on multiple displacement amplification in this application is also applicable to the kit for constructing a transcriptome library based on multiple displacement amplification, the spatial transcriptome library construction method based on multiple displacement amplification, the spatial transcriptome library construction method of biological samples, the transcriptome sequencing method of biological samples and the spatial transcriptome sequencing method of biological samples in the embodiments of this application, and will not be repeated here.

[0085] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0086] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0087] Example 1: Construction of a transcriptome library based on multiple displacement amplification

[0088] Experimental materials: Universal Human Reference RNA (Agilent), 100% methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), Nuclease-free Water (Invitrogen), 5× SSC (20× SSC stock solution (Invitrogen) formula: 3.0 M NaCl and 0.3 M sodium citrate, pH 7.0, diluted proportionally to 5×), 0.1× SSC (same as above, 20× SSC diluted proportionally to 0.1×), coverslips (Sita), 100% glycerol (Diomand), STOmics Permeabilization Kit-S1 (BGI), STOmics Library Preparation Kit-S1 (BGI), Stereo-seq Chip-P, MGISEQ-2000RS High-throughput Sequencing Kit PE100 (MGI), Klenow exo- DNA polymerase (BGI), MDA primer Random 1 (SEQ ID NO: 1, Sangon Biotechnology), XP magnetic beads (Agencourt), QubitTM ssDNA Assay Kit (Invitrogen), QubitTM dsDNA Assay Kit (Invitrogen),

[0089] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), MGISEQ-2000RS sequencer (MGI), 3.0 Fluorometer (Thermo Fisher)

[0090] Experimental methods:

[0091] mRNA was captured using a permeabilized microarray, and a transcriptome library was prepared using multiple displacement amplification with Random 1 primer.

[0092] Specifically, use the BGI Stereo-seq Chip-P, STOmics Transcriptome Reagent Set-S1 (reagent) and STOmics Library Construction Reagent Set-S1, refer to the "STOmics Stereo-seq Transcriptome Reagent Set User Manual" and "STOmics Stereo-seq Library Construction Reagent Set User Manual", and follow the following procedures:.

[0093] The specific process is as follows:

[0094] (1) Free RNA was hybridized with Chip P and incubated at room temperature for 5 min;

[0095] (2) Wash the chip with 0.1× SSC;

[0096] (3) Reverse transcription reaction, 42°C for 3 hours;

[0097] (4) Wash the chip with 0.1× SSC;

[0098] (5) Perform MDA reaction on the chip;

[0099] (6) Recover ssDNA using 1× magnetic beads;

[0100] (7) amplification of the recovered ssDNA by PCR;

[0101] (8) Magnetic bead double selection (0.55ⅹ+0.15ⅹ);

[0102] (9) DNBs were prepared and sequenced according to the MGISEQ-2000RS high-throughput sequencing kit PE100 (MGI) method, and the sequencing data were analyzed using a visual analysis system.

[0103] The mRNA hybridization system of step (1) is configured according to Table 2:

[0104] Table 2 Free RNA and permeabilized chip hybridization system

[0105] In step (3), the reverse transcription system uses the RT reagent in the STOmics Transcriptome Reagent Set-S1 (reagent);

[0106] In step (5), the chip MDA reaction was performed using Klenow exo- DNA polymerase.

[0107] MDA reaction system and process (Table 3):

[0108] Table 3 Chip MDA reaction system

[0109] Add the MDA reaction reagent into a PCR tube, and place the chip that has been hybridized with mRNA and completed the reverse transcription reaction into the PCR tube. First, perform MDA primer hybridization on a PCR instrument. The hybridization program is set according to Table 4.

[0110] Table 4 MDA primer hybridization reaction procedure

[0111] After the reaction, 5 μL of Klenow DNA polymerase (exo-) was added to the PCR tube, mixed, and incubated at 37°C for 1 hour.

[0112] Step (7) Using the reagents of the STOmics library preparation reagent set-S1, the ssDNA recovered from the 1x magnetic beads was subjected to library PCR and magnetic bead double selection according to the formula in Table 5 (step 8). After obtaining the library for the machine, DNB was made and the machine was sequenced (step 9).

[0113] Table 5 Library PCR system

[0114] Analysis of results: Sequencing results were analyzed to evaluate the mRNA capture of the library preparation process that includes MDA as a fragmentation step.

[0115] The basecall analysis of the original data was performed using the sequencer's built-in module, and sequencing indicators were output, including sequencing data volume, unique alignment rate, and duplicate alignment rate. The results are shown in Table 6.

[0116] Table 6 Sequencing performance of transcriptome libraries using MDA method

[0117] As can be seen from Table 6, the use of the MDA method for library construction achieved effective data output, indicating that the method of amplifying cDNA by MDA for fragmentation in the examples of the present application is feasible.

[0118] Example 2: Comparison of the effects of multiple displacement amplification and conventional Tn5 transposase disruption

[0119] This example compares a transcriptome library generated using MDA primers and multiple displacement amplification with a library generated using the existing Tn5 transposase fragmentation method to evaluate the effectiveness of the MDA library construction method proposed in this example. The specific experimental materials and equipment were essentially the same as in Example 1, with mouse brain tissue obtained from the Guangdong Medical Laboratory Animal Center.

[0120] Specifically, the MDA library construction method proposed in the examples of this application was used as the experimental group, and the existing Tn5 enzyme shearing library construction method was used as the control group. The control group used the BGI STOmics Gene Expression Reagent Set-S1 and the STOmics Library Construction Reagent Set-S1 and its standard operating procedure (including the Tn5 shearing step) to obtain the transcriptome library and sequence it; the experimental group used the PCR amplification product of the intermediate product cDNA of the control group as the starting DNA for MDA amplification, and used different MDA primers respectively. The library was constructed and sequenced according to the process described in Example 1, wherein Group 1 and Group 2 were set (corresponding to Random-1 and Random-2 as MDA primers, respectively, and their specific sequences are shown in Table 1).

[0121] The specific MDA database construction process is as follows:

[0122] (1) The PCR amplified product of the intermediate cDNA of the control group was prepared into an MDA system according to Table 7. The MDA process was referred to Table 4.

[0123] (2) recovering the ssDNA obtained in step (1) using 1x magnetic beads;

[0124] (3) Perform library PCR (prepared according to Table 5) and magnetic bead double selection to obtain the sequencing library;

[0125] (4) DNB preparation and sequencing, and data upload to the BGI STOmics visualization analysis system for analysis.

[0126] Table 7 Conventional MDA reaction system

[0127] Result analysis: Basecall analysis was performed on the raw data obtained from sequencing, and sequencing indicators were output: sequencing data volume, unique alignment rate, duplicate alignment rate, etc. The results are shown in Table 8.

[0128] Table 8 Sequencing performance of MDA library and Tn5 disruption library

[0129] As can be seen from Table 8, compared with the control group, at the same repeat alignment rate, the median Bin200MID of groups 1 and 2 reached or was close to that of the control group, and the median Bin200 genotype was close to or slightly higher than that of the control group. Both achieved capture levels comparable to or even better than those of the control group, indicating that the MDA-based library construction method proposed in the examples of this application can achieve high-quality and high-abundance mRNA capture, and effectively replace the traditional Tn5 enzymatic fragmentation method.

[0130] The above data demonstrate that the method for preparing transcriptome libraries based on MDA primers and multiple displacement amplification proposed in this example can effectively replace traditional library construction methods involving physical shearing (such as using ultrasound), shearing with a transposase (such as Tn5 enzyme), or in vitro transcription. While avoiding the problems of uneven shearing, easily degraded intermediates, and unstable library construction efficiency associated with traditional methods, it achieves data performance comparable to, and in some aspects superior to, those of traditional methods.

[0131] Example 3: Applicability test of MDA library construction for different tissues

[0132] This example uses mouse thymus tissue (from the Guangdong Medical Laboratory Animal Center) to further test the applicability and stability of transcriptome libraries prepared using MDA primers and multiple displacement amplification in different samples. Except for the use of mouse thymus tissue as the biological sample, all other experimental materials and equipment were the same as in Example 1.

[0133] Specifically, the MDA library construction method proposed in the examples of this application was used as the experimental group, and the existing Tn5 enzyme shearing library construction method was used as the control group. The control group used the BGI STOmics Gene Expression Reagent Set-S1 and the STOmics Library Construction Reagent Set-S1 and its standard operating procedure (which included a Tn5 shearing step) to obtain a transcriptome library based on mouse thymus tissue and sequence it; the experimental group used Random 1 as the MDA primer (see Table 1 for its specific sequence) and constructed and sequenced the mouse thymus tissue library according to the process described in Example 1 and the MDA process in Example 2.

[0134] Result analysis: Basecall analysis was performed on the raw data obtained from sequencing, and sequencing indicators were output: sequencing data volume, unique alignment rate, duplicate alignment rate, etc. The results are shown in Table 9.

[0135] Table 9 Sequencing data performance based on the transcriptome library of mouse thymus tissue

[0136] As shown in Table 9, the data quality obtained from sequencing the experimental group's libraries was comparable to that of the control group (Q30%: 87.22 vs 84.35). Furthermore, the experimental group demonstrated superior library data performance compared to the control group, with, for example, higher post-filter data volume (Clean Reads / Total Reads), more valid data volume (Valid CID Reads), and higher data abundance (Unique Reads). Furthermore, at similar duplication rates, the median capture of Bin200 genes obtained from MDA library construction was 4.92K, while that from Tn5 was 3.61K, demonstrating that the MDA method for library construction in this example outperformed the Tn5 fragmentation method in terms of capture capacity.

[0137] A heat map of the Bin50 MID medians for the two library construction methods is plotted, and the results are shown in Figure 3, where Figure 3(a) represents the control group and Figure 3(b) represents the experimental group. Comparing Figures 3a and 3b, the overall profiles and signal distributions of each component are roughly identical, demonstrating that the MDA library construction method of the present application achieves accurate mRNA signal detection consistent with the existing Tn5 interruption library construction method. Furthermore, under the same field of view parameters, the signal intensity in the visualization results of the MDA library construction method of the present application example is significantly stronger, which is consistent with the superior data performance presented in Table 9.

[0138] Bulk RNA from both groups was analyzed for genotype consistency, with the results shown in Figure 4. The left circle (blue) represents the results from the control group library, which yielded a total of 1,042 + 21,982 = 23,024 genes; the right circle (red) represents the results from the experimental group library, which yielded a total of 2,200 + 21,982 = 24,182 genes. As Figure 4 shows, the control and experimental group libraries capture a vast majority of the same genes (21,982 identical genes, representing 91% of the total genes detected in the experimental group library), demonstrating a high rate of gene overlap and minimal genotype differences between the two groups.

[0139] Pearson correlation analysis was performed on the bulk RNA genes and their expression levels from the two library construction methods. The results are shown in Figure 5. The vertical axis represents the experimental group, and the horizontal axis represents the control group. As shown in Figure 5, the correlation between the genes captured by the control and experimental libraries is extremely high (R = 0.9796).

[0140] The results shown in Figures 4 and 5 demonstrate that the MDA library construction method proposed in this example is similar to traditional techniques, achieving high-precision library construction and highly accurate mRNA information reduction based on this library. Furthermore, validation using mouse thymus tissue demonstrates that the MDA library construction method proposed in this example has good adaptability to different tissue types and can be widely used to construct libraries for different tissue and cell types.

[0141] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0142] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a transcriptome library based on multiple displacement amplification, comprising: Reverse transcribing mRNA molecules to obtain cDNA strands; Using the cDNA strands as templates, performing multiple displacement amplification under the action of MDA primers and a DNA polymerase with strand displacement activity to obtain multiple cDNA amplification fragments with different lengths, and the cDNA amplification fragments constitute the transcriptome library, wherein there are multiple MDA primers, and the MDA primers include a first sequence for sequencing and a second sequence for binding to the cDNA strands, the second sequence is a random sequence, and the second sequence randomly binds to the cDNA strands, Optionally, the first sequence is a first adapter, and the second sequence further includes degenerate bases.

2. The method according to claim 1, wherein the reverse transcription of mRNA molecules to obtain cDNA strands includes: Using the mRNA molecules as templates, performing reverse transcription under the action of reverse transcription primers and reverse transcriptase to obtain the cDNA strands, wherein the reverse transcription primers include a third sequence for capturing mRNA and an optional fourth sequence for labeling mRNA, Optionally, the third sequence is oligo dT (Oligo dT), and the fourth sequence is a unique molecular identifier sequence (UMI).

3. The method according to claim 2, wherein the reverse transcription primers further include a fifth sequence for sequencing, Optionally, the fifth sequence is a second adapter.

4. The method according to claim 2 or 3, wherein after obtaining multiple cDNA amplification fragments with different lengths, the method further includes: Based on the MDA primers or their fragments and optionally the reverse transcription primers or their fragments, performing secondary amplification on the multiple cDNA amplification fragments with different lengths.

5. The method according to any one of claims 1-4, after obtaining multiple cDNA amplification fragments with different lengths, the method further includes: Screening the multiple cDNA amplification fragments with different lengths to obtain the transcriptome library.

6. The method according to claim 5, wherein a solid-phase carrier is used for the screening to obtain cDNA amplification fragments with a desired length as the transcriptome library, Optionally, the solid-phase carrier is a silica-based magnetic bead, Optionally, the desired length is 200-800 nt, preferably 250-700 nt, more preferably 250-600 nt.

7. The method according to any one of claims 1 to 6, wherein after the reverse transcription and before the multiple displacement amplification, the method further comprises: Enriching the cDNA strands through a template switching mechanism.

8. The method according to claim 7, wherein enriching the cDNA strands through a template switching mechanism specifically includes: Adding a sixth sequence for template switching to the 3' end of the cDNA strands, optionally, the length of the sixth sequence is 1-20 nt, preferably 2-10 nt, optionally, the sixth sequence is polycytosine; Based on a template-switching oligonucleotide and a cDNA strand with the sixth sequence added at its end, an extended strand of the cDNA strand is obtained through template switching. Among them, the template-switching oligonucleotide contains a seventh sequence for binding to the sixth sequence. Optionally, the seventh sequence is polyguanine; and Amplify the extended strand to enrich the cDNA strand.

9. The method according to claim 8, wherein the template-switching oligonucleotide further contains an eighth sequence, where Based on the eighth sequence, amplify the extended strand to enrich the cDNA strand.

10. The method according to any one of claims 1 to 9, wherein the length of the first sequence in the MDA primer is 10 - 35 nt, preferably 10 - 25 nt, Optionally, the length of the second sequence is 4 - 15 nt, preferably 6 - 10 nt, and the proportion of degenerate bases in the second sequence is 100% or less, preferably higher than 75%, more preferably higher than 80%.

11. The method according to any one of claims 1 to 10 further includes: Based on the transcriptome library, prepare DNA nanoballs through rolling circle amplification.

12. The method according to any one of claims 1 to 11, wherein the DNA polymerase with strand displacement activity is selected from one or more of the following: Klenow DNA polymerase, reverse transcriptase, Phi29 DNA polymerase, Bst DNA polymerase, and Bsu DNA polymerase.

13. A kit for constructing a transcriptome library based on multiple displacement amplification, comprising: MDA reagents and reverse transcription reagents; And optionally fragment screening reagents and DNA nanoball preparation reagents, Wherein the MDA reagents include MDA primers and a DNA polymerase with strand displacement activity. The MDA primers contain a first sequence for sequencing and a second sequence for binding to the cDNA strand. The second sequence is a random sequence, and the second sequence randomly binds to the cDNA strand, Optionally, the first sequence is a first adapter, and the second sequence further contains degenerate bases.

14. The kit according to claim 13, wherein the length of the first sequence in the MDA primer is 10 - 35 nt, preferably 10 - 25 nt, Optionally, the length of the second sequence is 4 - 15 nt, preferably 6 - 10 nt, and the proportion of degenerate bases in the second sequence is 100% or less, preferably higher than 75%, more preferably higher than 80%, Preferably, the MDA primer is selected from one or two of SEQ ID NO: 1 - 2.

15. The kit according to claim 13 or 14, wherein the DNA polymerase with strand displacement activity is selected from one or more of the following: Klenow DNA polymerase, reverse transcriptase, Phi29 DNA polymerase, Bst DNA polymerase, and Bsu DNA polymerase.

16. The kit according to any one of claims 13-15, wherein the reverse transcription reagent comprises a reverse transcription primer, and the reverse transcription primer comprises a third sequence for capturing mRNA and optionally a fourth sequence for labeling mRNA. Optionally, the third sequence is Oligo dT, and the fourth sequence is a unique molecular identifier sequence (UMI). Preferably, the reverse transcription primer further comprises a fifth sequence for sequencing. Optionally, the fifth sequence is a second adapter.

17. The kit according to any one of claims 13-16, wherein the fragment screening reagent comprises a solid-phase carrier for screening cDNA amplification fragments to obtain cDNA amplification fragments with a desired length as the transcriptome library. Optionally, the solid-phase carrier is a silica-based magnetic bead. Optionally, the desired length is 200-800 nt, preferably 250-700 nt, more preferably 250-600 nt.

18. The kit according to any one of claims 13-17 further comprises a template switching reagent, wherein the template switching reagent comprises a template switching oligonucleotide, and the template switching oligonucleotide comprises a seventh sequence for binding to the 3'-end of the cDNA strand. Optionally, the seventh sequence is GGG. Optionally, the template switching oligonucleotide further comprises an eighth sequence for amplifying the extended strand.

19. A method for constructing a spatial transcriptome library of a biological sample, comprising: (1) In-situ capturing mRNA molecules from the biological sample; (2) Constructing a spatial transcriptome library according to the method for constructing a transcriptome library based on multiple displacement amplification according to any one of claims 1-12, wherein a spatial coordinate sequence for positioning the spatial position of the mRNA molecule in the biological sample is introduced into the cDNA strand to obtain a spatial transcriptome library. Optionally, the spatial coordinate sequence is introduced into the cDNA strand by making the reverse transcription primer comprise the spatial coordinate sequence. wherein the biological sample is a tissue section or a single-cell sample.

20. The method according to claim 19, wherein in-situ capturing mRNA molecules from the biological sample specifically comprises: Placing the biological sample on the surface of a capture chip; Fixing and permeabilizing the biological sample to release the mRNA molecules; and Using the capture chip to in-situ capture the mRNA molecules, wherein the capture chip is connected with the reverse transcription primer.

21. A method for transcriptome sequencing of a biological sample, comprising: Constructing a transcriptome library according to the method for constructing a transcriptome library based on multiple displacement amplification according to any one of claims 1-12; and Sequencing the transcriptome library to obtain the transcriptome data of the biological sample. Optionally, the biological sample is a tissue section or a single-cell sample.

22. A method for spatial transcriptome sequencing of a biological sample, comprising: Constructing a spatial transcriptome library according to the method for constructing a spatial transcriptome library based on multiple displacement amplification according to claim 19 or 20; and Sequencing the spatial transcriptome library to obtain the spatial transcriptome data of the biological sample, wherein according to the spatial coordinate sequence, the spatial position information of mRNA molecules in the biological sample is obtained. Optionally, the biological sample is a tissue section or a single-cell sample.

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