Single bacterium transcriptome sequencing method

Abstract

The invention discloses a single bacterium transcriptome sequencing method. The method comprises the following steps: firstly, mixing microorganisms and a wrapping material, solidifying to form a jelly-shaped semi-solid, and digesting cell walls by using lysozyme to permeate bacteria; adding a reverse transcription reagent and a random primer, and capturing total RNA in bacteria to synthesize cDNA; dissolving the wrapping material, releasing single bacteria, and packaging the single bacteria and the labeled microbeads into liquid drops by using a microfluidic device; rNA in bacteria is digested through enzyme digestion, so that cDNA is released from the bacteria, the cDNA is collected and purified after demulsification, a sequencing library is constructed through amplification and addition of a sequencing joint, a cDNA product of rRNA is digested, and a cDNA product of mRNA is enriched. And performing high-throughput sequencing on the processed sample, and analyzing transcriptome information of a single microorganism. Microorganisms are wrapped by the reversible solidification porous material, centrifugal cleaning is not needed, and single-bacterium analysis can be carried out on a microorganism sample with a small initial amount.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to transcriptome sequencing, and more specifically to a single bacterial transcriptome sequencing method. Background Art

[0002] The existing technology smRandom-seq (patent application number: CN202210174619.9) first fixes the isolated microbial samples, and fixes the bacteria with 4% paraformaldehyde overnight to cross-link the RNA, DNA and proteins inside the bacteria. Lysozyme is used to digest the cell wall and permeabilize the fixed bacteria for the next in situ reverse transcription reaction. The microorganism is used as a reaction vessel for in situ reaction, random primers are added to bind to the RNA in the bacteria, and the total RNA is captured by reverse transcription to synthesize cDNA, and the poly (dA) tail is added in situ to the 3' end of the cDNA by terminal transferase (TdT). After each step of the above process is completed, it is necessary to use buffer to wash about 3-5 times to prevent the influence of reagent residues on subsequent reactions. Single bacteria and labeled microbeads are encapsulated into droplets using a microfluidic device. The poly(T) primer is released from the microbeads by enzyme cleavage, and the RNA in the bacteria is digested to release the cDNA from the bacteria. The poly(T) primer binds to the poly(A) tail at the end of the cDNA, and then extends to add a specific code to the cDNA, and adds a molecular tag (UMI) to each cDNA. After demulsification, the cDNA is collected and purified, amplified and sequencing adapters are added to construct a sequencing library, and the cDNA product of rRNA is digested with cas9 to enrich the cDNA product of mRNA. The processed samples are subjected to high-throughput sequencing to analyze the transcriptome information of individual microorganisms.

[0003] Existing microbial single bacteria sequencing technologies: smRandom-seq (patent application number: CN202210174619.9); MicroSPLiT (microbial split-pool ligation transcriptomics, Kuchina A, BrettnerLM, Paleologu L, Roco CM, Rosenberg AB, Carignano A, et al. Microbial single-cell RNA sequencing by split-pool barcoding. Science 2021; 371(6531): eaba5257.); PETRI-seq (prokaryotic expression profiling by tagging RNA in situ and sequencing, Blattman SB, Jiang W, Oikonomou P, Tavazoie S. Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing. Nat Microbiol 2020; 5(10): 1192–201.).

[0004] Microorganisms are relatively small, and a high centrifugal speed (~4000g) is required during centrifugation. Multiple centrifugal washings can easily lead to the loss of microorganisms. At the same time, high-speed centrifugation can cause bacterial adhesion, and adherent microorganisms can cause double bacterial contamination. The existing technology has high requirements for the starting amount of microbial single bacterial analysis. The amount of microorganisms collected clinically is often relatively small, and culture amplification may affect the composition and state of the microorganisms, so microbial single bacterial analysis is often not possible.

[0005] PETRI-seq and MicroSPLIT are technologies based on combined indexing. After fixation and permeabilization of bacteria, reverse transcription (RT) is performed. In PETRI-seq, random primers are used for reverse transcription; in MicroSPLIT, the transcripts are first polyadenylated (poly(A) is added) before reverse transcription. RT is performed with ploy(T) primers. Both methods perform multiple rounds of index tag connection after RT (first distribute bacteria to different reaction wells and connect well-specific tags, then collect bacteria, mix and distribute to different reaction wells and connect well-specific tags, and repeat multiple times), connect specific sequence barcode tags to cDNA (barcode sequence is the position sequence of a certain bacterium in multiple rounds of distribution), and give each individual bacterium a unique identity. This process also has a large number of centrifugation and washing processes, including RT and each round of tag connection (2-3 rounds). There is a large requirement for the starting amount of samples (~10 million microorganisms), while the starting amount of the present invention is small (~100,000 microorganisms), which can meet more microbial single bacteria analysis scenarios. Summary of the invention

[0006] In view of the defects of the prior art, the present invention first fixes the microbial sample, and uses a reversible porous material (with reversible properties, which can be solid or liquid under different conditions; porous, with multiple pores inside the material, allowing biological macromolecules to pass through) to wrap the fixed bacterial sample. The material used for wrapping can be a natural biopolymer (such as at least one of agarose, gelatin, sodium alginate, and hyaluronic acid) or a synthetic material (such as at least one of polyethylene glycol and polyacrylamide). The microorganism and the wrapping material are mixed, placed in a mold, and solidified to form a jelly-like semisolid. There are pores in the middle of the wrapping material, which can allow macromolecular enzymes, salt ions, water, etc. to pass through, but can lock the microbial single bacteria in the wrapping material. After the wrapping is completed, the inclusion is transferred to a centrifuge tube or a special chip for cleaning and reaction. Since the microorganism has been locked in the inclusion, a centrifuge is not required for cleaning. Lysozyme is used to digest the cell wall and permeabilize the bacteria. Reverse transcription reagents and random primers are added to capture the total RNA in the bacteria to synthesize cDNA, and the poly(dA) tail is added in situ to the 3' end of the cDNA by terminal transferase (TdT). After each step of the above process, it is necessary to use buffer to wash about 3 times to prevent the influence of reagent residue on subsequent reactions. The encapsulation material is dissolved to release the single bacteria. After washing, the single bacteria and the labeled microbeads are encapsulated into droplets using a microfluidic device. The poly(T) primer is released from the microbeads by enzyme cleavage, and the RNA in the bacteria is digested to release the cDNA from the bacteria. The poly(T) primer binds to the poly(A) tail at the end of the cDNA, and then extends to add specific coding to the cDNA, and adds a molecular tag (UMI) to each cDNA. After demulsification, the cDNA is collected and purified, amplified and sequencing adapters are added to construct a sequencing library, and the cDNA product of rRNA is digested with cas9 to enrich the cDNA product of mRNA. The processed samples are subjected to high-throughput sequencing to analyze the transcriptome information of individual microorganisms.

[0007] The present invention provides a single bacterial transcriptome sequencing method, which comprises the following steps:

[0008] After fixing the microorganisms, they are wrapped with reversibly solidified porous materials to form inclusions, in which bacterial RNA is captured and linkers are added, and then the single bacteria are released. The processed samples are subjected to high-throughput sequencing to analyze the transcriptome information of individual microorganisms.

[0009] The wrapping material has pores in the middle, which allow macromolecular enzymes, salt ions and water to pass through, but the microorganisms are locked in the wrapping material.

[0010] Specifically, the steps include:

[0011] S1 fixes the microbial sample, and uses a reversible porous material to wrap the fixed bacterial sample, where the wrapping material is selected from natural biopolymers or artificial synthetic materials;

[0012] After S2 encapsulation is completed, the inclusions are washed, and lysozyme is used to digest the cell wall and permeabilize the bacteria;

[0013] S3 labels the RNA in the microorganisms, specifically, the inclusion sample is immersed in a labeling reagent, which includes a nucleotide chain, a tool enzyme and a reaction buffer, and the nucleotide chain is combined with the RNA fragment. After the nucleotide is combined with the RNA chain, a polymerization reaction or a connection reaction occurs, so that the nucleotide chain carries the RNA information and captures the connector; after completion, it is cleaned;

[0014] S4 dissolves the encapsulating material to release the single bacteria, and after washing, uses a microfluidic droplet or microplate system to segment the single bacteria to obtain a single droplet;

[0015] S5 performs an extension reaction on the single droplet after the single bacteria are segmented, breaks up the single droplet, purifies the cDNA, and performs PCR amplification;

[0016] S6 constructs a sequencing library and performs sequencing to analyze the transcriptome information of a single microorganism.

[0017] Preferably, the reversible porous material has a reversible property and can be solid or liquid under different conditions; and the reversible porous material has multiple pores inside, allowing biological macromolecules to pass through. Specifically, the natural biological macromolecule is selected from at least one of agarose, gelatin, sodium alginate, and hyaluronic acid; and the artificial synthetic material is selected from at least one of polyethylene glycol and polyacrylamide.

[0018] Further preferably, in step S3, the RNA of the microbial sample is labeled by reverse transcriptase, specifically, RNA labeling is performed by adding reverse transcriptase, nucleotide chain, and reaction buffer, and capture linker addition is performed by adding terminal transferase and a deoxyribonucleoside triphosphate.

[0019] In a specific embodiment, in step S4, the steps of using a microfluidic droplet system to perform single bacteria segmentation are as follows: connecting the microbial sample, the extension reaction reagent, the coded microsphere, and the oil phase to the corresponding liquid inlets of the microfluidic chip, respectively, to form a single oil-in-water droplet containing a single bacterium, a single coded microsphere, and an extension reaction reagent, and collecting the single droplets to form a single chamber containing a single bacterium;

[0020] In a specific embodiment, in step S5, the collected single droplet is subjected to an extension reaction to synthesize a second cDNA chain with a barcode label in the single droplet; after the extension reaction is completed, the single droplet is broken, and the cDNA in the extraction tube is purified and PCR amplification is performed;

[0021] In step S6, the cDNA amplified in step S5 is end-repaired and A-tailed using the TA cloning and ligation adapter library construction method, and the adapters are connected using a library construction kit to construct a sequencing library. The cDNA product of rRNA is digested with cas9, and the cDNA product of mRNA is enriched; the constructed library is subjected to high-throughput sequencing using the Illumina sequencing platform to analyze the transcriptome information of a single microorganism.

[0022] Preferably, the washing does not require centrifugation. Preferably, the washing is performed with a buffer solution to prevent the influence of reagent residues on subsequent reactions. The specific operation is to wash with PBST 2-4 times.

[0023] Optionally, the method further includes a data analysis step, specifically, clustering the detected microbial data.

[0024] Specifically, the microbial sample is an environmental microbial sample or a microbial sample in excrement or body fluid of an organism.

[0025] The advantages of the method of the present invention are as follows: the present invention wraps the microorganism with a reversible porous material to form an inclusion, converts centrifugal cleaning into non-centrifugal cleaning, and the microorganism completes the cleaning and RNA capture labeling reaction in the inclusion, thereby improving the recovery efficiency of the microorganism, reducing the adhesion ratio of the microorganism, and reducing the initial input amount of the microorganism, thereby realizing single bacterial analysis of a small amount (~100,000) of microbial samples. In addition, non-centrifugal cleaning is less difficult to achieve automation. The present invention first uses a reversible solidification porous material to wrap the microorganism before operating the microorganism, allowing the capture of bacterial RNA and the addition of a linker to be completed in the inclusion. Thereby, the centrifugal cleaning between reactions is converted into non-centrifugal cleaning, preventing microorganism agglomeration caused by high-speed centrifugation, reducing the loss of microorganisms caused by centrifugation, and reducing the change in bacterial ratio caused by centrifugation. The present invention can perform single bacterial analysis on microbial samples with a small initial amount. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Inclusion mold diagram.

[0027] Figure 2 Schematic diagram of the inclusion.

[0028] Figure 3 Results of staining of microorganisms in inclusions.

[0029] Figure 4 Agarose-encapsulated single bacterial microbial sequencing results.

[0030] Figure 5 Polyacrylamide encapsulated single bacterial microbial sequencing results.

[0031] Figure 6 Comparison of single bacteria recovery efficiency. DETAILED DESCRIPTION

[0032] The present invention is described below by means of specific examples to provide a better understanding of the present invention, but the present invention is not limited thereto.

[0033] Example 1: Low-melting-point agarose encapsulation of intestinal microbial samples for high-throughput single-bacterial transcriptome sequencing

[0034] Step 1: Microbial fixation: Take about 1 gram of fecal sample, add 5 ml of 4% PFA (paraformaldehyde), and mix by blowing. Place the mixture in a rotating mixer in a 4°C refrigerator and fix it overnight at 10-20 rpm for 12 hours. The fixed liquid is passed through a 40-micron cell sieve to remove larger impurities, collected in a 50-ml centrifuge tube, centrifuged at 500g, 4°C for 2 minutes, the supernatant is collected, passed through a 10-micron cell sieve, transferred to a 15-ml centrifuge tube, and centrifuged at 3900g, 4°C for 5 minutes to remove the fixative. Add 1 ml of PBS containing 1 microliter of RNase inhibitor, take 2 microliters of bacterial suspension sample, dilute with 998 microliters of water, stain with PI fluorescent dye and transfer to a slide, and observe the 200-fold field of view under a microscope for comparative evaluation of cell loss and morphological changes in subsequent experiments.

[0035] Step 2 Microbial encapsulation: Take 1 million bacteria, centrifuge at 3900g, 4°C, for 5 minutes, and remove the supernatant. Melt 1.5% low melting point agarose at 85°C and let it cool to room temperature. Take 50 μl of agarose to suspend the bacteria and transfer it to the solidification mold ( Figure 1 ) and let stand on ice for 15 minutes to solidify.

[0036] Step 3 Microbial permeabilization: Remove the inclusions from the mold. The inclusions are semi-solid and can be transferred to a 200 μL centrifuge tube using tweezers ( Figure 2 ), microorganisms are randomly distributed and embedded in inclusions ( Figure 3 ), soak in PBS containing RNaseinhibitor, and keep on ice for later use. Prepare cell wall digestion reagent (High-throughput intestinal flora single bacterial transcriptome kit from Hangzhou Yuezhen Biotechnology Co., Ltd.). Aspirate the PBS in the centrifuge tube, add 200 microliters of pre-cooled PBST and let stand on ice for 5 minutes for permeabilization. Add 800 microliters of PBS and centrifuge, aspirate the supernatant, and add 200 microliters of cell wall digestion solution. Place the centrifuge tube in a 37°C, 800rpm metal bath, react for 15 minutes, immediately take out and place on ice, aspirate the supernatant, add 200 microliters of pre-cooled PBST to terminate digestion, soak for 30 seconds, remove the supernatant, add 200 microliters of pre-cooled PBST and soak and wash twice, add 200 microliters of PBST and keep on ice for later use.

[0037] Step 4 RNA labeling: Prepare reverse transcription reagent (High-throughput intestinal flora single bacterial transcriptome kit of Hangzhou Yuezhen Biotechnology Co., Ltd.), 50 μl reverse transcription reagent (17.50 μl Buffer RN, 12.60 μl Buffer B2, 3.5 μl Buffer TX, 3.5 μl Enzyme R, 3.5 μl RNase inhibitor, 10 μl random reverse primer). Aspirate the supernatant in the centrifuge tube, add reverse transcription reagent, and place the centrifuge tube in a PCR instrument for gradient temperature cycling (8°C to 42°C, 11 cycles) reverse transcription reaction. After the reaction is completed, aspirate the supernatant, add 200 μl of washing solution to wash 3 times, and add 200 μl of washing solution on ice for later use. Prepare tailing reagent (High-throughput intestinal flora single bacterial transcriptome kit of Hangzhou Yuezhen Biotechnology Co., Ltd.), 50 μl A reagent (5 μl Buffer T1, 5 μl Buffer T2, 0.5 μl Adapter, 0.5 μl Enzyme T, 39 μl Buffer W). Aspirate the supernatant in the centrifuge tube, add reagent A, place the centrifuge tube in the PCR instrument, and incubate at 37°C for 30 minutes. After the reaction is completed, aspirate the supernatant and add 200 μl of cleaning solution to soak and wash 3 times.

[0038] Step 5: Dissolution of inclusions to release microorganisms: After washing, transfer the inclusions to a 1.5 ml centrifuge tube, place in a 65°C metal bath for 5 minutes, add 1 ml of washing solution, and mix by blowing. Centrifuge at 3900g, 4°C for 5 minutes and remove the supernatant. Take 2 μL of bacterial suspension and mix with 38 μL of water, then examine under a microscope, count and observe the bacterial status at this time.

[0039] Step 6 Microfluidic package: Configure microfluidic reagents (High-throughput intestinal flora single bacterial transcriptome kit from Hangzhou Yuezhen Biotechnology Co., Ltd.) and mix 100,000 bacteria (60 μl), 30 μl OptiPrep (sigma) and 30 μl extension reaction reagent to form a suspension on the machine. Prepare the microfluidic chip, add all 120 μl of the suspension on the machine to the suspension tank on the machine, add 55 μl of the labeled microspheres to the microsphere tank, and add the oil to the oil phase reservoir. After the sample is added, cover the chip with a sealing silicone pad. The sealing silicone pad should completely cover the upper surface of the chip, and the surface should be flat and without warping after covering. Place the chip in the VITacruizer single cell preparation instrument (Hangzhou Yuezhen Biotechnology Co., Ltd.) to generate microdroplets. Collect the droplets into a 200 μl centrifuge tube and place it in a PCR instrument for reaction (program: 37°C for 1 hour, 50°C for 30 minutes, 60°C for 30 minutes, 75°C for 20 minutes) to perform an extension reaction.

[0040] Step 7: Sequencing library construction: After the extension reaction is completed, PFO (perfluorooctane sulfonate) is added to the microdroplets. PFO releases the cDNA in the microemulsion droplets by destroying the surfactant molecular membrane of the oil phase shell. The cDNA is purified and extracted by the magnetic bead method. Part of the double-stranded cDNA is used as a template for qPCR experiments, and the total cDNA content captured is evaluated by the CT value. The remaining cDNA is amplified by PCR, and the amplified cDNA is end-repaired and A-tailed using the TA cloning connector library construction method, and the connector is connected using the library construction kit. After the constructed library, cas9 probes and enzymes are added to digest the cDNA products of rRNA, and high-throughput sequencing is performed using the Illumina sequencing platform.

[0041] Step 8 Data Analysis: The intestinal microbial data detected are as follows Figure 4 As shown in the figure, multiple microbial signals can be detected and clustered in fecal samples. The sequencing volume was 13.7G, and 4775 single bacteria were obtained, with an average of 562 reads per bacterium, an average UMI number of 257, and an average number of 38 genes.

[0042] Example 2: Encapsulation of microorganisms using polyacrylamide

[0043] The microorganisms were fixed according to step 1 of Example 1.

[0044] Step 2 Microbial encapsulation: Prepare acrylamide materials and buffer according to the paper Dissolvable Polyacrylamide Beads for High-Throughput Droplet DNA Barcoding (AdvancedScience 2020). Add 50 μL TBSET buffer, 30 μL 10% (w / v) APS (Sigma-Aldrich, A9164), 75 μL 40% (v / v) Acrylamide solution (Sigma-Aldrich, A4058-100ML), 245 μL 0.8% (w / v) BAC (Sigma-Aldrich, A4929-5G) and 100 μL double distilled water to a centrifuge tube, mix well, take 50 μL, suspend 1 million bacteria, add 0.5ul TEMED, and mix well. Place in a mold and solidify at 40°C for 30 minutes.

[0045] According to step 3 of Example 1, microorganism permeabilization was performed, and according to step 4, RNA labeling was performed.

[0046] Step 5: Dissolution of inclusions to release microorganisms: After the reaction is completed, the inclusions are washed and placed in a centrifuge tube. 500 μL of 1 mmol DTT is added to dissolve the inclusions. 1 ml of washing solution is added and mixed by pipetting. Centrifuge at 3900g, 4°C for 5 minutes to remove the supernatant. Take 2 μL of bacterial suspension and mix with 38 μL of water and examine under a microscope to count and observe the bacterial status at this time.

[0047] Microfluidic packaging was performed according to step 6 of Example 1, and sequencing library construction was performed according to step 7.

[0048] Step 8 Data Analysis: The intestinal microbial data detected are as follows Figure 5 As shown in the figure, multiple microbial signals can be detected and clustered in fecal samples. The sequencing volume was 9.5G, and 3098 single bacteria were obtained, with an average of 386 reads per bacterium, an average UMI number of 180, and an average number of 21 genes.

[0049] Example 3: Comparison of bacterial recovery rates between the present invention and smRandom-seq

[0050] The microbial samples were prepared and counted using the smRandom-seq method (Droplet-based high-throughput single microbe RNAsequencing by smRandom-seq) published in Nature communications in 2023. Compared with the recovery efficiency of the microbial samples in Examples 1 and 2, the recovery efficiency of the microorganisms was significantly improved after encapsulation ( Figure 6 ). In the smRandom-seq method, a large number of microbial samples are centrifuged and washed, and about 60% of the bacteria are retained after RT, and only about 40% of the bacteria are retained after adding A tail. With the method of the present invention, about 90% of the bacteria can still be recovered after encapsulation, washing and adding A tail, which reduces the number of starting microorganisms for analysis.

Claims

1. A single bacterial transcriptome sequencing method, characterized in that: The steps include: After fixing the microorganisms, they are wrapped with reversibly solidified porous materials to form inclusions, in which bacterial RNA is captured and linkers are added, and then the single bacteria are released. The processed samples are subjected to high-throughput sequencing to analyze the transcriptome information of individual microorganisms. The wrapping material has pores in the middle, which allow macromolecular enzymes, salt ions and water to pass through, but the microorganisms are locked in the wrapping material.

2. The single bacterial transcriptome sequencing method according to claim 1, characterized in that: The steps include: S1 fixes the microbial sample, and uses a reversible porous material to wrap the fixed bacterial sample, where the wrapping material is selected from natural biopolymers or artificial synthetic materials; After S2 encapsulation is completed, the inclusions are washed, and lysozyme is used to digest the cell wall and permeabilize the bacteria; S3 labels the RNA in the microorganisms, specifically, the inclusion sample is immersed in a labeling reagent, which includes a nucleotide chain, a tool enzyme and a reaction buffer, and the nucleotide chain is combined with the RNA fragment. After the nucleotide is combined with the RNA chain, a polymerization reaction or a connection reaction occurs, so that the nucleotide chain carries the RNA information and captures the connector; after completion, it is cleaned; S4 dissolves the encapsulating material to release the single bacteria, and after washing, uses a microfluidic droplet or microplate system to segment the single bacteria to obtain a single droplet; S5 performs an extension reaction on the single droplet after the single bacteria are segmented, breaks up the single droplet, purifies the cDNA, and performs PCR amplification; S6 constructs a sequencing library and performs sequencing to analyze the transcriptome information of a single microorganism.

3. The single bacterial transcriptome sequencing method according to claim 2, characterized in that: The reversible porous material has a reversible property and can be in a solid state or a liquid state under different conditions; and the reversible porous material has multiple pores inside, allowing biological macromolecules to pass through.

4. The single bacterial transcriptome sequencing method according to claim 2, characterized in that: The natural biopolymer is selected from at least one of agarose, gelatin, sodium alginate and hyaluronic acid; the artificial synthetic material is selected from at least one of polyethylene glycol and polyacrylamide.

5. The single bacterial transcriptome sequencing method according to claim 2, characterized in that: In step S3, the RNA of the microbial sample is labeled by reverse transcriptase, specifically, RNA labeling is performed by adding reverse transcriptase, nucleotide chain, and reaction buffer, and capture linker is added by adding terminal transferase and a deoxyribonucleoside triphosphate.

6. The single bacterial transcriptome sequencing method according to claim 2, characterized in that: In step S4, the steps of using a microfluidic droplet system to perform single bacteria segmentation are as follows: the microbial sample, extension reaction reagent, coded microsphere, and oil phase are respectively connected to the corresponding liquid inlets of the microfluidic chip to form a single oil-in-water droplet containing a single bacterium, a single coded microsphere, and an extension reaction reagent, and the single droplets are collected to form a single chamber containing a single bacterium.

7. The single bacterial transcriptome sequencing method according to claim 2, characterized in that: In step S5, the collected single droplet is subjected to an extension reaction to synthesize a second cDNA chain with a barcode in the single droplet; after the extension reaction is completed, the single droplet is broken, and the cDNA in the extraction tube is purified and PCR amplification is performed; In step S6, the cDNA amplified in step S5 is end-repaired and A-tailed using the TA cloning and ligation adapter library construction method, and the adapters are connected using a library construction kit to construct a sequencing library. The cDNA product of rRNA is digested with cas9, and the cDNA product of mRNA is enriched; the constructed library is subjected to high-throughput sequencing using the Illumina sequencing platform to analyze the transcriptome information of a single microorganism.

8. The single bacterial transcriptome sequencing method according to claim 2, characterized in that: The washing does not require centrifugation. Preferably, the washing is performed with a buffer solution to prevent the influence of reagent residues on subsequent reactions. The specific operation is to wash with PBST 2-4 times.

9. The single bacterial transcriptome sequencing method according to claim 7, characterized in that: The method also includes a data analysis step, specifically clustering the detected microbial data.

10. The single bacterial transcriptome sequencing method according to any one of claims 1 to 9, characterized in that: The microbial sample is an environmental microbial sample or a microbial sample in excrement or body fluid of an organism.

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