Space-time transcriptome sequencing method, sequencing library construction method and gene sequencing system

Abstract

The invention discloses a space-time transcriptome sequencing method, a sequencing library construction method and a gene sequencing system. The space-time transcriptome sequencing method comprises the following steps that 1, the 3'end of a probe sequence and the 3 'end of template RNA are complementarily combined for reverse transcription, a template conversion oligonucleotide sequence is added to the tail end of generated cDNA through reverse transcriptase, and the template conversion oligonucleotide sequence is different from a first connector sequence at the 5' end of the probe sequence; 2) amplifying the cDNA by using the first linker sequence and the template conversion oligonucleotide sequence; and (3) sequencing the cDNA amplified in the step (2). By utilizing the method disclosed by the invention, the time for sequencing the space-time transcriptome can be shortened, and the 5'end sequencing of the space-time transcriptome can be realized.

Description

Spatiotemporal transcriptome sequencing methods, sequencing library construction methods, and gene sequencing systems Technical Field

[0001] The present invention relates to the field of gene sequencing technology, and in particular to a spatiotemporal transcriptome sequencing method, a sequencing library construction method and a gene sequencing system. Background Art

[0002] The spatiotemporal transcriptome sequencing technology developed by BGI is an ultra-high-throughput and ultra-high-resolution spatiotemporal transcriptome technology. This technology utilizes spatiotemporal microarrays to capture mRNA in tissues in situ, enabling the localization and differentiation of active expression of functional genes within specific tissue regions. Furthermore, this technology can also achieve in situ transcriptome capture of single cells. As the fundamental unit of the organism, cells collaborate with their microenvironment in specific spatial locations to exert their unique biological functions. Therefore, spatial location information is particularly important for studying and understanding the mechanisms of cell biology, tumor biology, developmental biology, and other disciplines. The development of spatiotemporal transcriptome technology provides important reference value for developmental research and disease diagnosis.

[0003] Template Switch Oligo (TSO) is an oligonucleotide that hybridizes to the non-template C nucleotide added by the reverse transcriptase during reverse transcription. In existing spatiotemporal transcriptome sequencing technologies, the TSO sequence added by the reverse transcriptase during reverse transcription is consistent with the Read1 adapter sequence on the chip. Therefore, after cDNA amplification and purification, the fragments with identical sequences at both ends are fragmented using a transposase to prepare the library for sequencing. The principle is that the transposase can cut double-stranded DNA (dsDNA) and connect the resulting DNA ends to the Read2 adapter, thereby achieving sequencing.

[0004] Existing spatiotemporal transcriptome sequencing technologies are time-consuming and cannot achieve 5′-end sequencing of the spatiotemporal transcriptome, and thus cannot achieve TCR / BCR sequencing in spatiotemporal transcriptome-tumor immunology research.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a spatiotemporal transcriptome sequencing method, a sequencing library construction method and a gene sequencing system.

[0007] Therefore, in a first aspect, the present invention provides a spatiotemporal transcriptome sequencing method, comprising the following steps: 1) performing reverse transcription by complementary binding between the 3′ end of the probe sequence and the 3′ end of the template RNA, and adding a template conversion oligonucleotide sequence to the end of the generated cDNA using a reverse transcriptase, wherein the template conversion oligonucleotide sequence is different from the first linker sequence at the 5′ end of the probe sequence; 2) amplifying the cDNA using the first linker sequence and the template conversion oligonucleotide sequence; and 3) sequencing the cDNA amplified in step 2).

[0008] Furthermore, the method further includes tissue permeabilization before step 1).

[0009] Furthermore, the 3' end of the probe sequence is a poly-T structure, and the 3' end of the template RNA is a poly-A structure.

[0010] Furthermore, step 3) includes: circularizing the cDNA amplified in step 2), preparing DNA nanoballs, and sequencing the DNA nanoballs.

[0011] Furthermore, the probe sequence includes a coordinate identifier sequence and a molecular identifier sequence.

[0012] Furthermore, the probe sequence is on a gene chip.

[0013] Furthermore, the template switching oligonucleotide sequence is 5′-GAGACGTTCTCGACTCAGCAGA / rG / / rG / / iXNA_G / .

[0014] In a second aspect, the present invention provides a method for constructing a sequencing library, comprising the following steps: 1) performing reverse transcription by complementary binding between the 3′ end of the probe sequence and the 3′ end of the template RNA, and adding a template conversion oligonucleotide sequence to the end of the generated cDNA using a reverse transcriptase, wherein the template conversion oligonucleotide sequence is different from the first adapter sequence at the 5′ end of the probe sequence; and 2) amplifying the cDNA using the first adapter sequence and the template conversion oligonucleotide sequence.

[0015] Furthermore, the method further includes tissue permeabilization before step 1).

[0016] Furthermore, the 3' end of the probe sequence is a poly-T structure, and the 3' end of the template RNA is a poly-A structure.

[0017] Furthermore, the method further comprises step 3): forming a circle of the cDNA amplified in step 2) to prepare DNA nanoballs.

[0018] Furthermore, the probe sequence includes a coordinate identifier sequence and a molecular identifier sequence.

[0019] Furthermore, the probe sequence is on a gene chip.

[0020] Furthermore, the template switching oligonucleotide sequence is 5′-GAGACGTTCTCGACTCAGCAGA / rG / / rG / / iXNA_G / .

[0021] In a third aspect, the present invention provides a gene sequencing system, which utilizes the spatiotemporal transcriptome sequencing method described in the first aspect or the sequencing library construction method described in the second aspect.

[0022] By adopting the above scheme, the beneficial effects of the present invention are: shortening the time of spatiotemporal transcriptome technology to 2 days; and realizing the 5′ end sequencing technology of spatiotemporal transcriptome. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a schematic diagram showing the principle of the spatiotemporal transcriptome sequencing technology in an embodiment of the present invention.

[0024] FIG2 shows the 2100 detection results of the TSO-1 and TSO-2 solutions in an embodiment of the present invention.

[0025] FIG3 is a visual comparison data of the TSO-1 and TSO-2 solutions in an embodiment of the present invention.

[0026] FIG4 is a clustering consistency analysis of adjacent slices of the TSO-1 and TSO-2 schemes in an embodiment of the present invention.

[0027] FIG5 is a 5′ end coverage analysis of the TSO-1 and TSO-2 schemes in the examples of the present invention, TSS: Transcription Start Site, TES: Transcription End Site, Transcription Termination Site. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, the scientific and technical terms used in this application have the meanings commonly understood by those skilled in the art. In order to better understand the present invention, the following definitions and explanations of relevant terms are provided.

[0030] In this article, a template switch oligonucleotide (TSO) is an oligonucleotide that hybridizes to a non-template C nucleotide added by the reverse transcriptase during reverse transcription. The simplest TSO has three riboguanosines (rGrGrG) at its 3' end. The complementarity between these consecutive rG bases and the 3'dC stretch of the cDNA molecule enables subsequent template switching.

[0031] In this article, the coordinate identifier (CID) refers to a spatial coordinate barcode. For example, in the Stereo-seq technology, a random 25nt sequence is synthesized as the CID, and the spatial position of the mRNA molecule is restored by the CID sequence after sequencing.

[0032] Herein, a molecular identifier (MID), also referred to as a unique molecular identifier (UMI), is used to distinguish transcripts. For example, in Stereo-seq technology, the MID and an oligonucleotide containing a polyT sequence are linked to the CID by hybridization.

[0033] In this article, DNB (DNA Nanoball) refers to DNA nanoballs. In DNB sequencing, after the genomic DNA fragments are circularized into single-stranded circular DNA, the circular single-stranded DNA is formed into multiple copies of single-stranded DNA connected end to end through rolling circle amplification technology, and freely folded into a nanoball structure in the solution, namely DNA nanoball (DNB).

[0034] As used herein, the term "primer" refers to an isolated nucleic acid molecule that anneals to a complementary target DNA chain through nucleic acid hybridization, forms a hybrid between the primer and the target DNA chain, and then extends along the target DNA chain under the action of a polymerase (e.g., DNA polymerase).

[0035] It should also be noted that, in this document, relational terms such as first and second, etc., are used only 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. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or terminal device.

[0036] As mentioned above, the present invention aims to provide a spatiotemporal transcriptome sequencing method, a sequencing library construction method and a gene sequencing system.

[0037] In the first aspect, the present invention provides a spatiotemporal transcriptome sequencing method, comprising the following steps: 1) performing reverse transcription by complementary binding between the 3′ end of the probe sequence and the 3′ end of the template RNA, and adding a template conversion oligonucleotide sequence to the end of the generated cDNA using a reverse transcriptase, wherein the template conversion oligonucleotide sequence is different from the first linker sequence at the 5′ end of the probe sequence; 2) amplifying the cDNA using the first linker sequence and the template conversion oligonucleotide sequence; and 3) sequencing the cDNA amplified in step 2).

[0038] In one embodiment, the method further comprises tissue permeabilization before step 1).

[0039] In one embodiment, the 3' end of the probe sequence is a poly-T structure, and the 3' end of the template RNA is a poly-A structure.

[0040] In one embodiment, step 3) comprises: circularizing the cDNA amplified in step 2), preparing DNA nanoballs, and sequencing the DNA nanoballs.

[0041] The probe sequence includes CID and MID, where CID (Coordinate Identity) is the coordinate identifier and MID (Molecular Identifier) ​​is the molecular identifier. The CID and MID on each probe are different, thereby achieving specific recognition sequence. The above probe sequence is on the gene chip.

[0042] In one embodiment, the TSO sequence is 5'-GAGACGTTCTCGACTCAGCAGA / rG / / rG / / iXNA_G / (SEQ ID NO: 1).

[0043] In a second aspect, the present invention provides a method for constructing a sequencing library, comprising the following steps: 1) performing reverse transcription by complementary binding between the 3′ end of the probe sequence and the 3′ end of the template RNA, and adding a template conversion oligonucleotide sequence to the end of the generated cDNA using a reverse transcriptase, wherein the template conversion oligonucleotide sequence is different from the first adapter sequence at the 5′ end of the probe sequence; and 2) amplifying the cDNA using the first adapter sequence and the template conversion oligonucleotide sequence.

[0044] In one embodiment, the method further comprises tissue permeabilization before step 1).

[0045] In one embodiment, the 3' end of the probe sequence is a poly-T structure, and the 3' end of the template RNA is a poly-A structure.

[0046] In one embodiment, the method further comprises step 3): circularizing the cDNA amplified in step 2) to prepare DNA nanoballs.

[0047] In one embodiment, the probe sequence comprises a coordinate identifier sequence and a molecular identifier sequence.

[0048] In one embodiment, the probe sequences are on a gene chip.

[0049] In one embodiment, the template switching oligonucleotide sequence is 5'-GAGACGTTCTCGACTCAGCAGA / rG / / rG / / iXNA_G / (SEQ ID NO: 1).

[0050] In a third aspect, the present invention provides a gene sequencing system, which utilizes the spatiotemporal transcriptome sequencing method described in the first aspect or the sequencing library construction method described in the second aspect.

[0051] Example

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0053] The principle diagram of the spatiotemporal transcriptome sequencing technology of this embodiment is shown in Figure 1, which specifically includes the following steps: First, the sample tissue is permeabilized; the 3' end poly A of the sample RNA is complementary to the poly T of the probe on the chip, which also includes the first adapter (Read1), CID, and MID; the reverse transcription reaction begins under the action of reverse transcriptase to generate cDNA; cDNA is amplified to obtain a library. The primer sequences in this embodiment that are inconsistent with existing spatiotemporal transcriptome sequencing technologies are as follows:

[0054] The sequence of TSO-2 is 5′-GAGACGTTCTCGACTCAGCAGA / rG / / rG / / iXNA_G / (SEQ ID NO: 1).

[0055] The sequence of cDNA-F-pho is 5′-CTGCTGACGTACTGAGAGGC (SEQ ID NO: 2), wherein 5′-pho is 5′ phosphorylated and modified. This sequence is used as an upstream primer for cDNA amplification.

[0056] The sequence of cDNA-R is 5'-GAGACGTTCTCGACTCAGCAGA (SEQ ID NO: 3), which was used as a downstream primer for cDNA amplification.

[0057] The sequence of MDA was 5′-CCCTCTGCTGAGTCGAGAACGTCTC (SEQ ID NO: 4), which was the primer sequence used for sequencing.

[0058] After amplification, TSO-2 becomes the second linker sequence (Read2), the sequence of which is 5′-GAGACGTTCTCGACTCAGCAGAGGG (SEQ ID NO: 5).

[0059] The experimental steps are as follows:

[0060] 1) Frozen mouse brain tissue was obtained, sliced ​​into 10 μm sections, and mounted on the spatiotemporal chip. The sections were baked at 37°C for 3 minutes and then fixed in methanol at -20°C for 30 minutes.

[0061] 2) Add 100 μL of ssDNA for staining, incubate at room temperature for 5 min, aspirate and discard the liquid, and wash once with 0.1× SSC;

[0062] 3) Cover the slides with 5 μL of glycerol, photograph the FITC channel to collect fluorescence images, and wash the glycerol by soaking the 24-well plate in 500 μL of 5× SSC for 5 minutes;

[0063] 4) Remove the chip and wipe the liquid on the back of the chip, aspirate the liquid on the front, add 100 μL of 1× tissue permeabilization enzyme, and permeabilize at 37°C for 12 minutes. After permeabilization, aspirate and discard, and wash once with 0.1× SSC;

[0064] 5) Prepare the reverse transcription reaction solution according to Table 1, with a final TSO-2 concentration of 2.5 μM. Add 100 μL of the reverse transcription reaction solution dropwise and incubate at 42°C for 2 h.

[0065] Table 1: Reverse transcription reaction mixture

[0066] Components Single reaction volume RT Reagent 80μL RT Additive 5μL RI 5μL Reverse T Enzyme 5μL TSO-2 (50μM) 5μL Total 100μL

[0067] 6) Add 400 μL tissue removal solution, incubate at 55°C for 5 min, remove the tissue removal solution, and wash once with 0.1× SSC;

[0068] 7) Add 400 μL cDNA release solution and incubate at 55°C for 3 h;

[0069] 8) Purify the cDNA release solution with 0.8× magnetic beads, and use 42 μL to dissolve it back. Prepare the PCR reaction solution according to Table 2 and perform the PCR reaction.

[0070] Table 2: PCR reaction mixture

[0071] Components Single Reaction Volume Recovery Sample 42 μL 2× High-Fidelity Ready Mix 50 μL cDNA-F-pho (10 μM) 4 μL cDNA-R (10 μM) 4 μL Total 100 μL

[0072] 9) Purify the PCR product using 0.8× magnetic beads and store frozen at -20°C;

[0073] 10) Direct circularization, DNB preparation, and sequencing;

[0074] 11) Data analysis and visualization.

[0075] Experimental results:

[0076] Four consecutive slices were made: two slices were made using the original technical solution TSO-1; the other two slices were made using the TSO-2 designed in the present invention.

[0077] The 2100 detection results are shown in FIG2 . The cDNA fragment distributions obtained using the TSO-2 of the present invention and the original technology are consistent. As shown in Table 3 , the cDNA yields obtained by 0.8× purification are similar, with no significant difference.

[0078] Table 3: 0.8× cDNA yield

[0079]

[0080] The visualization results are shown in FIG3 . The number of genes and other technical indicators in the visualization results using TSO-2 of the present invention and the original technology are consistent, and the performance is consistent within the repeated groups using the technology of the present invention, indicating that the technology of the present invention is stable.

[0081] As shown in Figure 4, after performing de-batch clustering analysis on the data and restoring spatial location information, the clustering results and restored spatial location information using the TSO-2 method are completely consistent with those of the original method. A1, A2, A4, and A5 in Figure 4 correspond to the results of four slices, respectively, and the results show a high degree of cluster information overlap.

[0082] As shown in FIG5 , the data were analyzed for transcriptome 5′ end coverage: the original technical solution lost the 5′ end during the disruption by Tn5 transposase, and therefore had no 5′ end information; the information obtained using the technology of the present invention contained obvious 5′ end information, and thus spatiotemporal transcriptome 5′ end sequencing could be achieved.

[0083] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations are readily apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A spatiotemporal transcriptome sequencing method, characterized in that: The method comprises the following steps: 1) Reverse transcription is performed by complementary binding between the 3′ end of the probe sequence and the 3′ end of the template RNA, and a template switching oligonucleotide sequence is added to the end of the generated cDNA using a reverse transcriptase, wherein the template switching oligonucleotide sequence is different from the first adapter sequence at the 5′ end of the probe sequence; 2) amplifying the cDNA using the first linker sequence and the template switching oligonucleotide sequence; 3) sequencing the cDNA amplified in step 2).

2. The spatiotemporal transcriptome sequencing method according to claim 1, characterized in that: The method further includes tissue permeabilization before step 1).

3. The spatiotemporal transcriptome sequencing method according to claim 1, characterized in that: The 3' end of the probe sequence is a poly-T structure, and the 3' end of the template RNA is a poly-A structure.

4. The spatiotemporal transcriptome sequencing method according to claim 1, characterized in that: Step 3) includes: circularizing the cDNA amplified in step 2), preparing DNA nanoballs, and sequencing the DNA nanoballs.

5. The spatiotemporal transcriptome sequencing method according to claim 1, characterized in that: The probe sequence includes a coordinate identifier sequence and a molecular identifier sequence.

6. The spatiotemporal transcriptome sequencing method according to claim 1, characterized in that: The probe sequence is on a gene chip.

7. The spatiotemporal transcriptome sequencing method according to claim 1, characterized in that: The template switching oligonucleotide sequence is 5′-GAGACGTTCTCGACTCAGCAGA / rG / / rG / / iXNA_G / .

8. A method for constructing a sequencing library, characterized in that: The method comprises the following steps: 1) performing reverse transcription by complementary binding between the 3′ end of the probe sequence and the 3′ end of the template RNA, and adding a template conversion oligonucleotide sequence to the end of the generated cDNA using a reverse transcriptase, wherein the template conversion oligonucleotide sequence is different from the first adapter sequence at the 5′ end of the probe sequence; and 2) amplifying the cDNA using the first adapter sequence and the template conversion oligonucleotide sequence.

9. The method for constructing a sequencing library according to claim 8, characterized in that: The method further includes tissue permeabilization before step 1).

10. The method for constructing a sequencing library according to claim 8, characterized in that: The 3' end of the probe sequence is a poly-T structure, and the 3' end of the template RNA is a poly-A structure.

11. The method for constructing a sequencing library according to claim 8, characterized in that: The method further comprises step 3): forming a circle of the cDNA amplified in step 2) to prepare DNA nanoballs.

12. The method for constructing a sequencing library according to claim 8, characterized in that: The probe sequence includes a coordinate identifier sequence and a molecular identifier sequence.

13. The method for constructing a sequencing library according to claim 8, characterized in that: The probe sequence is on a gene chip.

14. The method for constructing a sequencing library according to claim 8, characterized in that: The template switching oligonucleotide sequence is 5′-GAGACGTTCTCGACTCAGCAGA / rG / / rG / / iXNA_G / .

15. A gene sequencing system, characterized in that: The gene sequencing system utilizes the spatiotemporal transcriptome sequencing method described in any one of claims 1 to 7 or the sequencing library construction method described in any one of claims 8 to 14.

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