Heterologous target sequence specific enrichment method based on space-time transcription technology and application

Abstract

The invention relates to the technical field of space-time transcriptomes, in particular to a space-time transcription technology-based heterologous target sequence specific enrichment method and application. The invention provides a heterologous target sequence specific enrichment method based on a space-time transcription technology and application. In the reaction process of space-time transcriptome analysis, the method provided by the invention can be used for specifically enriching interested specific sequences (heterologous target sequences) while space-time gene information of the whole tissue slice is reserved, so that the relationship between heterologous genes and gene expression modes in tissues can be better researched. According to the method provided by the invention, an enrichment process based on combination of space transcriptome and multi-omics analysis is perfected, and a reliable basis is provided for multi-omics research, for example, bacterial distribution information in a host, infection mode information of the host, local response of the host and the like can be obtained from a bacterial infected tissue sample.

Description

Technical Field

[0001] The present invention relates to the field of spatiotemporal transcriptome technology, and in particular to a method and application of heterologous target sequence specific enrichment based on spatiotemporal transcription technology. Background Art

[0002] Spatiotemporal transcriptomics refers to the use of high-throughput sequencing technology to study gene expression patterns in tissues or cells with temporal and spatial resolution. Compared with traditional transcriptomics technology, spatiotemporal transcriptomics technology can provide more detailed temporal and spatial information, revealing differences in gene expression patterns at different times and in different regions within tissues and cells. This technology can help scientists better understand the temporal and spatial distribution and interactions of genes in organisms, thereby deeply exploring complex issues such as cell types, tissue structures, and the development and function of organisms.

[0003] Multiplex PCR is a PCR technique that simultaneously amplifies multiple target sequences in a single reaction. In traditional PCR, usually only one target sequence can be amplified. However, multiplex PCR uses multiple primers to amplify multiple target sequences, and multiple genes or DNA fragments can be detected and amplified simultaneously in the same reaction. This technology can greatly improve the efficiency of PCR and save samples, and is particularly suitable for situations where multiple targets need to be detected simultaneously, such as pathogen detection, genotyping, DNA fingerprinting, etc. The key to multiplex PCR is to design appropriate primers, each primer corresponds to a target sequence, and their specificity cannot interfere with each other. By optimizing primer design and reaction conditions, multiple target sequences can be amplified simultaneously, thereby improving experimental efficiency and accuracy. Multiplex PCR technology has been widely used in medical diagnosis, genotyping, disease research and other fields.

[0004] Nested PCR is an improved PCR technique that aims to improve the specificity and sensitivity of PCR. In nested PCR, two rounds of PCR reactions are connected in series, and the first round of PCR products serve as templates for the second round of PCR. This nested PCR design can improve the specificity of PCR because the internal primers are located inside the target sequence. Only when the first round of PCR successfully amplifies the target sequence will sufficient templates be generated for the second round of PCR. In addition, since the primers of the second round of PCR are closer to the target sequence, nested PCR can also improve the sensitivity of PCR, and the target sequence can be effectively amplified even if it is only present at very low concentrations.

[0005] At present, multi-omics research based on spatiotemporal transcriptome is developing rapidly. By integrating spatiotemporal transcriptome data with other multi-omics data, we can have a more comprehensive understanding of the relationship between molecular composition and function in organisms, providing powerful tools and methods for revealing the complexity of biological systems. This comprehensive research method is expected to provide new ideas and strategies for biomedical research and drug development. Microbiome is a discipline that studies the composition, structure, function of microbial communities and their interactions with hosts and the environment. Microbiome uses high-throughput sequencing technology and bioinformatics methods to comprehensively analyze and study the species, abundance, metabolic functions, etc. of microorganisms in microbial communities. Among them, bacterial 16S identification is a commonly used microbial identification method based on sequence analysis of the 16S rRNA gene. The 16S rRNA gene exists in the ribosomal RNA of bacteria and archaea and is a conserved ribosomal RNA gene in bacterial cells. The gene is highly conserved and variable. The conserved region is used to design universal primers, and the variable region is used for species identification. There are few joint studies on spatiotemporal transcriptomics and microbiome, and no mature application solutions have been proposed yet.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] To solve the above technical problems, the present invention provides a method and application of heterologous target sequence specific enrichment based on spatiotemporal transcription technology, which can help us more comprehensively understand the interaction between microorganisms and hosts, especially in complex ecosystems, such as the interaction between intestinal microorganisms and host intestinal mucosa.

[0008] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology, comprising the following steps: S1, total cDNA obtained by reverse transcription; S2, performing a round of multiplex amplification using the total cDNA as a template; the primers for the multiplex amplification include homologous target sequence primers and heterologous target sequence primers; S3. Divide the product of one round of multiple amplification into multiple parts, and perform two rounds of nested amplification on each part; the primers for the two rounds of nested amplification are selected from homologous target sequence primers or heterologous target sequence primers.

[0009] Preferably, the reverse transcription in step S1 is performed on spatiotemporal transcriptome microspheres; and the spatiotemporal transcriptome microspheres are connected to capture primers of heterologous target sequences.

[0010] Preferably, the source of the heterologous target sequence is a microorganism.

[0011] Preferably, the microorganism is selected from bacteria and viruses.

[0012] Preferably, the capture primer is selected from at least one of SEQ ID NO.01, SEQ ID NO.02 and SEQ ID NO.03.

[0013] Preferably, there are two heterologous target sequence primers, and their nucleotide sequences are shown as SEQ ID NO.4 and SEQ ID NO.6 respectively.

[0014] Preferably, there are two homologous target sequence primers, and their nucleotide sequences are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0015] In a second aspect, the present invention provides the application of the heterologous target sequence specific enrichment method based on spatiotemporal transcription technology in studying the interaction between microorganisms and hosts (such as the interaction between intestinal microorganisms and host intestinal mucosa).

[0016] It should be noted that the application of the present invention is not intended for the diagnosis or treatment of a disease. If the application of the present invention involves the diagnosis or treatment of a disease, the technical solution in this case is not within the scope of protection of the present invention.

[0017] Beneficial effects: The present invention provides a method and application for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology. In the reaction process of spatiotemporal transcriptome analysis, the method provided by the present invention can be used to specifically enrich specific sequences of interest (heterologous target sequences) while retaining the spatiotemporal gene information of the entire tissue section, thereby better studying the relationship between heterologous genes and gene expression patterns in tissues. The method provided by the present invention improves the enrichment process based on spatial transcriptome combined with multi-omics analysis, and provides a reliable foundation for multi-omics research. For example, for bacterially infected tissue samples, information on bacterial distribution in the host and its infection pattern, local responses of the host, etc. can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0019] Figure 1 Schematic diagram of the positions of capture probes 1 to 3 and enrichment primers in the present invention.

[0020] Figure 2This is the 5400 quality inspection image of the cDNA (bacterial enrichment product) of bact (bacteria) corresponding to the cpA (capture A) microspheres in the present invention.

[0021] Figure 3 This is the 5400 quality inspection image of the cDNA (bacterial enrichment product) of bact (bacteria) corresponding to the cpB (capture B) microspheres in the present invention.

[0022] Figure 4 This is the 5400 quality inspection image of the bact (bacteria) cDNA (bacterial enrichment product) corresponding to the cpC (capture C) microspheres in the present invention.

[0023] Figure 5 This is the 5400 quality inspection image of the host cDNA (host enrichment product) corresponding to the cpA (capture A) microspheres in the present invention.

[0024] Figure 6 This is the 5400 quality inspection image of the host cDNA (host enrichment product) corresponding to the cpB (capture B) microspheres in the present invention.

[0025] Figure 7 This is the 5400 quality inspection image of the host cDNA (host enrichment product) corresponding to the cpC (capture C) microspheres in the present invention.

[0026] Figure 8 It is the fragment length distribution diagram of bact (bacteria) cDNA of cpA (capture A) microspheres in the present invention before nanopore data filtering and the corresponding mapped fragment length distribution diagram aligned to the reference genome.

[0027] Fig. 9 It is the fragment length distribution diagram of bact (bacteria) cDNA of cpB (capture B) microspheres in the present invention before nanopore data filtering and the corresponding mapped fragment length distribution diagram aligned to the reference genome.

[0028] Fig.10 It is the fragment length distribution diagram of bact (bacteria) cDNA of cpC (capture C) microspheres in the present invention before nanopore data filtering and the corresponding mapped fragment length distribution diagram aligned to the reference genome.

[0029] Fig.11 This is a technical flow diagram of the solution of Example 1 of the present invention. DETAILED DESCRIPTION

[0030] The purpose of the present invention is to build a general method for specific enrichment of heterologous target sequences based on spatiotemporal transcriptome. In order to verify this method, the present invention enriched the bacterial 16S target region of the product on the microspheres of the Biomarker microbiome combined spatial transcriptome chip. The fragment distribution was detected by Agilent 5400, and the target sequence was compared by high-throughput sequencing, and the results were stable and reliable.

[0031] The specific implementation plan is as follows: (1) Primer design: The present invention uses three capture primers targeting different variable regions of bacterial 16S, namely: capture probe 1~3 (abbreviated as cp1~3) and a specific enrichment primer, the position of which is shown in Figure 1 .

[0032] (2) Microsphere synthesis: cp1~3 were connected to the Biotron S1000 spatial transcriptome microspheres at a ratio of 50%, and the three groups of microspheres were named cpA~C.

[0033] (3) Reverse transcription on microspheres: RNA (human enterobacterium: Enterobacterium = 1:1) and reverse transcription-related reagents were added to the three groups of microspheres and incubated at 42°C with shaking for 1.5 h.

[0034] (4) Second-chain synthesis on microspheres: After reverse transcription, the microspheres were treated with KOH to release the RNA strands, and then DNA polymerase was used to synthesize the second strand.

[0035] (5) One round of multiplex amplification of cDNA: Use KOH to dissolve the second strand on the microsphere and add Tris-HCL (pH=7), then add two sets of primers, ssp / Read1 and Enrichment primer / Read1, at a concentration of 10uM, and amplify for 5 cycles.

[0036] (6) Second round of cDNA nested amplification: The product after one round of amplification was purified using 0.8× and divided into two parts. The first part was amplified using 10uM ssp / Read1 for 8 cycles, and the second part was amplified using Enrichment primer / Read1 for 15 cycles.

[0037] (7) Purification and quality inspection of cDNA products: After the cycle, the product was purified (0.8×), the first aliquot was named host, and the second aliquot was named bact. The fragment distribution quality inspection of the purified product was performed using 5400. The results are shown in Figure 2-7 .

[0038] (8) Third-generation library construction and sequencing: Use the corresponding Naopore kit to construct the third-generation library of the bact product and sequence it.

[0039] (9) Third-generation data analysis: Download the 16S database from the blast database on the NCBI website, align the sequences to the corresponding database, screen the blast results, and retain the results with pident*length greater than 70% of the sequence length and aligned to the target species. Use R language to perform length distribution on the retained sequences. The results are shown in Figure 8-10 .

[0040] The universal method for specific enrichment of target sequences based on spatial transcriptome provided by the present invention has the advantages of simple operation (only one additional round of PCR is performed on the Biochuang S1000 spatial transcriptome process) and stable results (three different capture primers can obtain fragments and data of corresponding lengths).

[0041] In this invention, the study of spatial transcriptome combined with microbiome is taken as an example. The core is to build a method for specific enrichment of microorganisms such as bacterial sequences by means of multiple PCR and nested PCR. This method is suitable for specific enrichment of microbial target sequences based on spatiotemporal transcriptomes. It is a general method and is not limited to the cases listed.

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used can be obtained from commercial sources.

[0043] Example 1 This embodiment provides a general method for specific enrichment of target sequences based on spatial transcriptome, comprising the following steps: 1. Primer Design The primer sequences involved in this embodiment are shown in Table 1 below (where ssp and Read1 are primers known and used in the art).

[0044] Table 1 2. Microsphere Synthesis Based on the microspheres on the currently commercialized Biochuang S1000 spatial transcriptome chip, cp1~3 were connected to the microspheres through linkers to synthesize microspheres cpA, cpB and cpC.

[0045] 3. Reverse transcription on microspheres 3.1. Divide cpA, cpB and cpC microspheres into three groups and wash them once with enzyme-free water.

[0046] 3.2. Add the reverse transcription premix as shown in Table 2 below to each group of microspheres (the reagents are from Biotron S1000 Gene Expression V3.1 Kit).

[0047] Table 2 3.3. After mixing by pipetting, place the cells in a shaking incubator at 42°C, 90 min, 2000 rpm.

[0048] 4. Second-chain synthesis on microspheres 4.1. After reverse transcription, centrifuge briefly and discard the supernatant.

[0049] 4.2. Add 20ul 0.08M KOH and mix thoroughly by pipetting, then incubate at room temperature for 5min.

[0050] 4.3. Centrifuge briefly, discard the supernatant, add 50ul EB, mix by pipetting, and incubate at room temperature for 2 minutes.

[0051] 4.4. Centrifuge briefly, discard the supernatant, and add the second-strand synthesis premix as shown in Table 3 (reagents are from Biochuang S1000 Gene Expression V3.1 Kit).

[0052] Table 3 4.5. After mixing by pipetting, place the cells in a shaking incubator at 65°C, 2000 rpm for 30 min.

[0053] 5. One round of multiplex amplification of cDNA 5.1. After the second-strand synthesis is completed, centrifuge immediately, discard the supernatant, add 50ul EB, pipette and mix, and incubate at room temperature for 2 minutes.

[0054] 5.2. Centrifuge briefly, discard the supernatant, add 23ul 0.08M KOH, mix by pipetting, and incubate at room temperature for 10min.

[0055] 5.3. Centrifuge briefly, pipette 21ul of supernatant into a new tube, add 3ul of Tris-HCL (pH=7), and mix thoroughly by pipetting.

[0056] 5.4. So far, the second-strand products of three groups of microspheres were obtained. A round of multiplex amplification system was prepared according to Table 4 below (Amp Mix was from Biotron S1000 Gene Expression V3.1 Kit).

[0057] Table 4 5.5. Perform amplification according to the procedure shown in Table 5 below.

[0058] Table 5 6. cDNA second round nested amplification 6.1. Use SPRI magnetic beads to perform 0.8× purification on the products of one round of multiplex amplification, and finally use 46ul enzyme-free water for resolubilization.

[0059] 6.2. Prepare the host and bacteria two-round nested amplification systems according to Tables 6 and 7 below.

[0060] Table 6 Host two-round nested amplification system Table 7 Bacteria (bact) two-round nested amplification system 6.3. Perform amplification according to Table 8 below.

[0061] Table 8 7. Purification and quality inspection of cDNA products 7.1. Purify the products of the second round of amplification using SPRI magnetic beads (0.8×).

[0062] 7.2. After measuring the concentration, use Agilent 5400 for quality inspection.

[0063] 8. Construction and sequencing of third-generation libraries The nanopore kit was used to obtain the third-generation library according to the process of final repair, barcoding, library mixing and adapter addition, and the Nanopore Flow cell R9 was used for the machine.

[0064] 9. Three-generation data analysis The 16S database was downloaded from the blast database of the NCBI website, and the sequences were aligned to the corresponding database. The blast results were screened, and the results with pident*length greater than 70% of the sequence length and aligned to the target species were retained. 10,000 reads were uniformly extracted and the analysis data are shown in Tables 9 and 10 below.

[0065] Table 9 Note: (1) mapped: the number of reads aligned to the bacterial reference genome. (2) primer mapped: the number of reads that can be blasted to the enrichment primer in the mapped reads.

[0066] Table 10 Note: (1) mapped: the number of reads aligned to the human reference genome. (2) primer mapped: the number of reads that can be blasted to ssp in the mapped reads.

[0067] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solution of the present invention in detail, but cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.

Claims

1. A method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology, characterized in that: The steps include: S1, total cDNA obtained by reverse transcription; S2, performing a round of multiplex amplification using the total cDNA as a template; the primers for the round of multiplex amplification include homologous target sequence primers and heterologous target sequence primers; S3. Divide the multiple amplification products of one round into multiple parts, and perform two rounds of nested amplification on each part; the primers for the two rounds of nested amplification are selected from homologous target sequence primers or heterologous target sequence primers.

2. According to claim 1, the method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology is characterized in that: The reverse transcription in step S1 is performed on the spatiotemporal transcriptome microspheres; the spatiotemporal transcriptome microspheres are connected to capture primers of heterologous target sequences.

3. The method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology according to claim 1 or 2, characterized in that: The source of the heterologous target sequence is a microorganism.

4. The method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology according to claim 3, characterized in that: The microorganism is selected from bacteria and viruses.

5. The method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology according to claim 4, characterized in that: The capture primer is selected from at least one of SEQ ID NO.01, SEQ ID NO.02 and SEQ ID NO.

03.

6. The method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology according to claim 4, characterized in that: There are two heterologous target sequence primers, and their nucleotide sequences are shown as SEQ ID NO.4 and SEQ ID NO.6 respectively.

7. The method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology according to claim 4, characterized in that: There are two homologous target sequence primers, and their nucleotide sequences are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

8. Application of the method for specific enrichment of heterologous target sequences based on spatiotemporal transcription technology according to any one of claims 1 to 7 in studying the interaction between microorganisms and hosts.

Images