CN119487209A

CN119487209A - Spatiotemporal transcriptome sequencing method, sequencing library construction method and gene sequencing system

Info

Publication number: CN119487209A
Application number: CN202280097967.9A
Authority: CN
Inventors: 廖莎; 陈奥; 章文蔚; 徐讯; 周慧; 郭晶; 杨晶
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2025-02-18
Also published as: WO2024138690A1

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 of the present invention comprises the steps of 1) performing reverse transcription by complementarily binding the 3' -end of a probe sequence to the 3' -end of a template RNA, and adding a template switching oligonucleotide sequence to the end of the generated cDNA by using reverse transcriptase, wherein the template switching oligonucleotide sequence is different from a first linker sequence at the 5' -end of the probe sequence, 2) amplifying the cDNA by using the first linker sequence and the template switching oligonucleotide sequence, and 3) sequencing the cDNA amplified in the step 2). The method can shorten the time for sequencing the space-time transcriptome and realize the 5' end sequencing of the space-time transcriptome.

Description

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

Technical Field

The invention relates to the technical field of gene sequencing, in particular to a space-time transcriptome sequencing method, a sequencing library construction method and a gene sequencing system.

Background

The space-time transcriptome sequencing technology for Huada gene development is a super-high flux and super-high resolution space-time transcriptome technology. The technology utilizes a space-time chip to capture mRNA in tissues in situ, and can locate and distinguish active expression of functional genes in specific tissue areas. In addition, the technology can also realize single cell in situ transcriptome capture. The cell is taken as a basic unit of an organism, and cooperates with the microenvironment at a specific spatial position to play a special biological function, so that the spatial position information is particularly important for researching and understanding the generation mechanism of subjects such as cell biology, tumor biology, developmental biology and the like, and the development of space-time transcriptome technology provides important reference value for development research and disease diagnosis.

A template switch oligonucleotide (TEMPLATE SWITCH Oligo, TSO) is an oligonucleotide that hybridizes to a non-template C nucleotide that is added by a reverse transcriptase during reverse transcription. In the existing space-time transcriptome sequencing technology, the TSO sequence added by reverse transcriptase is consistent with the Read1 joint sequence on a chip during reverse transcription, so that after cDNA amplification and purification, fragments with consistent sequences at both ends need to be interrupted by using transposase for library construction sequencing, and the principle is that the transposase can cut double-stranded DNA (dsDNA) and connect the tail end of the obtained DNA with the Read2 joint, thereby realizing sequencing.

The existing space-time transcriptome sequencing technology is long in time consumption, and can not realize 5' end sequencing of space-time transcriptomes, so that TCR/BCR sequencing can not be realized in space-time transcriptome-tumor immunology research.

Disclosure of Invention

The invention aims to provide a space-time transcriptome sequencing method, a sequencing library construction method and a gene sequencing system.

Thus, in a first aspect, the invention provides a method of spatiotemporal transcriptome sequencing comprising the steps of 1) reverse transcription with complementary binding of the 3' end of a probe sequence to the 3' end of a template RNA and addition of a template switching oligonucleotide sequence to the end of the generated cDNA using a reverse transcriptase, said template switching oligonucleotide sequence being different from the first adaptor sequence at the 5' end of said probe sequence, 2) amplification of said cDNA using said first adaptor sequence and said template switching oligonucleotide sequence, 3) sequencing of said cDNA amplified in step 2).

Further, the method comprises tissue permeabilization prior to step 1).

Further, 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.

Further, step 3) includes looping the cDNA amplified in step 2), preparing a DNA nanosphere, and sequencing the DNA nanosphere.

Further, the probe sequence includes a coordinate identifier sequence and a molecular identifier sequence.

Further, the probe sequence is on a gene chip.

Further, the template switch oligonucleotide sequence is 5' -GAGACGTTCTCGACTCAGCAGA/rG// rG// iXNA _G/.

In a second aspect, the present invention provides a method of constructing a sequencing library, the method comprising the steps of 1) reverse transcription with complementary binding of the 3' end of a probe sequence to the 3' end of a template RNA, and adding a template switching oligonucleotide sequence to the end of the generated cDNA using reverse transcriptase, the template switching oligonucleotide sequence being different from a first linker sequence at the 5' end of the probe sequence, and 2) amplification of the cDNA using the first linker sequence and the template switching oligonucleotide sequence.

Further, the method comprises tissue permeabilization prior to step 1).

Further, the method further comprises 3) a step of making the cDNA amplified in 2) loop to prepare a DNA nanosphere.

Further, the probe sequence is on a gene chip.

In a third aspect, the present invention provides a gene sequencing system using the spatiotemporal transcriptome sequencing method of the first aspect or the sequencing library construction method of the second aspect.

By adopting the scheme, the method has the beneficial effects of shortening the time of the space-time transcriptome technology to 2 days and realizing the 5' end sequencing technology of the space-time transcriptome.

Drawings

FIG. 1 is a schematic diagram of a space-time transcriptome sequencing technique in an embodiment of the invention.

FIG. 2 shows the results of 2100 measurements of the TSO-1 and TSO-2 schemes in examples of the present invention.

FIG. 3 is a visual comparison of TSO-1 and TSO-2 schemes in an embodiment of the present invention.

FIG. 4 is an adjacent slice cluster consistency analysis of TSO-1 and TSO-2 schemes in an embodiment of the present invention.

FIG. 5 is a 5' end coverage analysis of TSO-1 and TSO-2 schemes, TSS: transcription START SITE, transcription initiation site, TES: transcription End Site, transcription termination site, in examples of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise defined, scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art. For a better understanding of the present application, definitions and explanations of the relevant terms are provided below.

In this context, a template switch oligonucleotide (TEMPLATE SWITCH Oligo, TSO) is an oligonucleotide that hybridizes to a non-template C nucleotide that is added by a reverse transcriptase during reverse transcription. The simplest TSO has 3 riboguanines (rGrGrG) at its 3 'end, and the complementarity between these consecutive rG bases and the 3' dC extension of the cDNA molecule enables subsequent template conversion.

In this context, the coordinate identifier (Coordinate Identity, CID) refers to the spatial coordinate barcode, e.g., a random 25nt sequence is synthesized as CID in the Stereo-seq technique, and the spatial position of the mRNA molecule is reduced by the CID sequence after sequencing.

In this context, molecular identifiers (Molecular Identifier, MID) are also referred to as unique molecular markers (Unique Molecular Identifier, UMI) for distinguishing transcripts, for example, MID and oligonucleotides containing polyT sequences are linked to CID by hybridization in the Stereo-seq technique.

In this context DNB (DNA Nanoball) refers to DNA nanospheres, in DNB sequencing, after circularization of the genomic DNA fragments into single-stranded circular DNA, the circular single-stranded DNA is formed into multiple copies of single-stranded DNA by end-to-end by rolling circle amplification techniques and is free to fold into a nanosphere structure in solution, i.e. DNA Nanospheres (DNB).

As used herein, the term "primer" refers to an isolated nucleic acid molecule that binds to a complementary strand of target DNA by nucleic acid hybridization, anneals to form a hybrid between the primer and the strand of target DNA, and then extends along the strand of target DNA under the action of a polymerase (e.g., DNA polymerase).

It is further noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal.

As previously mentioned, the present invention is directed to a space-time transcriptome sequencing method, a method of constructing a sequencing library, and a gene sequencing system.

In a first aspect, the invention provides a space-time transcriptome sequencing method comprising the steps of 1) reverse transcription with complementary binding of the 3' end of a probe sequence to the 3' end of a template RNA and adding a template switching oligonucleotide sequence to the end of the generated cDNA using a reverse transcriptase, said template switching oligonucleotide sequence being different from a first linker sequence at the 5' end of said probe sequence, 2) amplification of said cDNA using said first linker sequence and said template switching oligonucleotide sequence, 3) sequencing of said cDNA amplified in step 2).

In one embodiment, the method further comprises tissue permeabilization prior to step 1).

In one embodiment, the probe sequence is poly-T in 3 'and the template RNA is poly-a in 3' end.

In one embodiment, step 3) comprises looping the cDNA amplified in step 2), preparing a DNA nanosphere, and sequencing the DNA nanosphere.

The probe sequences include CID and MID, wherein CID (Coordinate Identity) is a coordinate identifier, MID (Molecular Identifier) is a molecular identifier, and CID and MID on each probe are different, thereby realizing a specific recognition sequence. The probe sequence is on a gene chip.

In one embodiment, the TSO sequence is 5' -GAGACGTTCTCGACTCAGCAGA/rG// rG// iXNA _G/(SEQ ID NO: 1).

In a second aspect, the present invention provides a method of constructing a sequencing library comprising the steps of 1) reverse transcription with complementary binding of the 3' end of a probe sequence to the 3' end of a template RNA, and adding a template switching oligonucleotide sequence to the end of the generated cDNA using reverse transcriptase, said template switching oligonucleotide sequence being different from a first adaptor sequence at the 5' end of said probe sequence, and 2) amplification of said cDNA using said first adaptor sequence and said template switching oligonucleotide sequence.

In one embodiment, the method further comprises 3) a step of making the cDNA amplified in 2) loop to prepare a DNA nanosphere.

In one embodiment, the probe sequence includes a coordinate identifier sequence and a molecular identifier sequence.

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

In one embodiment, the template switch oligonucleotide sequence is 5' -GAGACGTTCTCGACTCAGCAGA/rG// rG// iXNA _G/(SEQ ID NO: 1).

Examples

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The principle schematic diagram of the space-time transcriptome sequencing technology in the embodiment is shown in fig. 1, and specifically comprises the following steps of firstly, carrying out tissue permeabilization on a sample, complementarily combining poly-A at the 3' -end of RNA of the sample with poly-T of a probe on a chip, wherein the chip also comprises a first connector (Read 1), CID and MID, starting reverse transcription reaction under the action of reverse transcriptase to generate cDNA, and amplifying the cDNA to obtain a library. The primer sequences in this example that are inconsistent with existing space-time transcriptome sequencing techniques are as follows:

the sequence of TSO-2 is 5' -GAGACGTTCTCGACTCAGCAGA/rG// rG// iXNA _G/(SEQ ID NO: 1).

The sequence of cDNA-F-pho is 5' -CTGCTGACGTACTGAGAGGC (SEQ ID NO: 2), wherein 5' -pho is a 5' phosphorylation modification, which acts as an upstream primer in cDNA amplification.

The sequence of cDNA-R was 5' -GAGACGTTCTCGACTCAGCAGA (SEQ ID NO: 3) as a downstream primer in cDNA amplification.

The sequence of the sequenced MDA is 5' -CCCTCTGCTGAGTCGAGAACGTCTC (SEQ ID NO: 4), which is the primer sequence used for sequencing.

TSO-2 is amplified to become a second linker sequence (Read 2) which is 5' -GAGACGTTCTCGACTCAGCAGAGGG (SEQ ID NO: 5).

The experimental procedure was as follows:

1) Taking frozen brain tissue of a mouse, slicing the frozen brain tissue into 10 mu m slices, attaching the slices on a space-time chip, baking the slices at 37 ℃ for 3min, and fixing methanol at-20 ℃ for 30min;

2) 100. Mu.L of ssDNA is dripped for dyeing, the room temperature is 5min, the liquid is sucked and removed, and the solution is washed for 1 time by 0.1 XSSC;

3) 5 mu L of glycerol seal, taking a photograph by a FITC channel, collecting fluorescent images, soaking the fluorescent images in 500 mu L of 5 XSSC for 5min by using a 24-pore plate, and cleaning the glycerol;

4) Taking out the chip, wiping the liquid on the back of the chip, sucking the liquid on the front, dripping 100 mu L of 1 XSSC, permeabilizing for 12min at 37 ℃, sucking and discarding after permeabilization is finished, and cleaning for 1 time by 0.1 XSSC;

5) Preparing a reverse transcription reaction solution according to Table 1, wherein the final concentration of TSO-2 is 2.5. Mu.M, dropwise adding 100. Mu.L of the reverse transcription reaction solution, and standing at 42 ℃ for 2 hours;

TABLE 1 reverse transcription reaction solution

Component (A)	Single reaction volume
RT Reagent	80μL
RT Additive	5μL
RI	5μL
Reverse T Enzyme	5μL
TSO-2(50μM)	5μL
Total	100μL

6) Adding 400. Mu.L of tissue removing solution, removing the tissue removing solution at 55 ℃ for 5min, and washing with 0.1 XSSC for 1 time;

7) 400. Mu.L of cDNA releasing solution was added at 55℃for 3 hours;

8) The cDNA release solution was purified using 0.8X1 beads, and 42. Mu.L of the solution was reconstituted, and PCR reaction was performed by preparing a PCR reaction solution according to Table 2;

TABLE 2 PCR reaction solution

Component (A)	Single reaction volume
Recovering the sample	42μL
2×High-Fidelity Ready Mix	50μL
cDNA-F-pho(10μM)	4μL
cDNA-R(10μM)	4μL
Total	100μL

9) 0.8Xmagnetic bead purified PCR product, -20 ℃ frozen stock;

10 Direct cyclization, DNB preparation, and on-machine sequencing;

11 Data analysis and visualization.

Experimental results:

4 continuous slices were made, 2 slices using the original technical scheme TSO-1 and 2 additional slices using the TSO-2 designed in the present invention.

2100 Detection results are shown in FIG. 2, and cDNA fragments obtained by using the TSO-2 of the present invention and the original technology are distributed consistently, and as shown in Table 3, yields of cDNA obtained by 0.8Xpurification are close, without significant differences.

TABLE 3 0.8 XcDNA yield

The visual results are shown in fig. 3, and the base factors and other technical indexes in the visual results of the TSO-2 and the prior art are consistent in performance, and the repeated group of the technology is consistent in performance, so that the technical performance of the invention is stable.

As shown in FIG. 4, the data is subjected to batch removal clustering analysis, and the spatial position information is restored, and the clustering result and the restored spatial position information of the TSO-2 are completely consistent with the prior art. The results of the four slices corresponding to A1, A2, A4 and A5 in fig. 4 show that the overlapping degree of the clustering information is high.

As shown in FIG. 5, the transcriptome 5' coverage analysis of the data is performed, in which the original technical scheme loses the 5' end when Tn5 transposase is interrupted, so that the 5' end information is absent, and the information obtained by the technology of the invention has obvious 5' end information, so that the space-time transcriptome 5' end sequencing can be realized.

The above is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

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).

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

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.

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

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.

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

The spatiotemporal transcriptome sequencing method according to claim 1, characterized in that the template switching oligonucleotide sequence is

5′-GAGACGTTCTCGACTCAGCAGA/rG//rG//iXNA_G/.

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 a probe sequence and the 3′ end of a 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.

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

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.

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

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.

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

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/.

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 method for constructing a sequencing library described in any one of claims 8 to 14.