Method for efficiently capturing conjugate plasmid group

Abstract

The invention discloses a method for efficiently capturing a conjugate plasmid group. According to the method, a filter membrane bonding method is combined with selective sequencing, a large number of bonding plasmids can be obtained at a time, the high efficiency of capturing the bonding plasmids by a complex environment sample is improved, the capturing efficiency of the bonding plasmids is remarkably improved, and the excellent capturing capacity for a large-scale bonding plasmid group is shown. The method is suitable for research on the conjugate plasmid group of the microbial community in the complex environment, does not need a plasmid extraction kit, is simpler and more convenient, and has lower preference. And the applicability and the performance are high.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and particularly to a method for efficiently capturing conjugative plasmidomes. Background Art

[0002] Conjugative plasmids are important genetic elements carrying horizontally transferable functional genes (such as antibiotic resistance genes) and are key vectors for gene dissemination in the environment. In complex ecosystems, conjugative plasmids are widely spread among different species through conjugation, and have important ecological and public health significance in the processes of microbial evolution, diffusion, and enrichment of resistance genes. A conjugative plasmidome refers to the collection of all conjugative plasmids in an environmental sample, a single microorganism, or a specific microbial community. Comprehensive and systematic research on it has important value for understanding the mechanism of horizontal gene transfer and the occurrence and dissemination patterns of resistance genes in the environment. Currently, the research on conjugative plasmidomes still faces multiple technical bottlenecks. The filter mating method is one of the common molecular biology techniques for capturing conjugative plasmids from environmental samples. Although this method has achieved targeted enrichment of conjugative plasmids with drug resistance or specific functions in the environment at the experimental level, it still has limitations such as low throughput, low efficiency, and high sequencing costs, and it is difficult to meet the systematic analysis requirements for the diversity of environmental conjugative plasmidomes, community occurrence patterns, and transfer rules.

[0003] On the other hand, bioinformatics prediction methods based on metagenomics have become an important technical path for current plasmidome research. This method usually performs de novo assembly on the community metagenome to obtain genomic fragments or contigs, and combines with public plasmid databases (such as PLSDB) for sequence alignment, or uses machine learning models (such as Plasflow) for prediction to identify potential plasmid sequences. Although this method has the advantages of high throughput and wide coverage, it essentially relies on computational prediction and lacks experimental conjugation verification, making it difficult to accurately distinguish free, functional plasmids with conjugative transfer ability. At the same time, due to the extremely low proportion of plasmid DNA in the community metagenome (usually less than 0.1%), conventional assembly methods are difficult to achieve complete reconstruction of plasmids, and are prone to problems such as fragmentation, false positives, and false assembly, seriously affecting the accuracy and systematicness of the research.

[0004] Therefore, there is an urgent need in the prior art for a method for capturing and assembling conjugative plasmids with high selectivity, high throughput, and high accuracy, so as to improve the breadth and depth of conjugative plasmidome research and provide more solid data support for the study of resistance dissemination rules and ecological risk assessment. Summary of the Invention

[0005] Aiming at the defects in the prior art, the present invention proposes a method for efficiently capturing conjugative plasmidomes.

[0006] The present invention provides a method for efficiently capturing a conjugative plasmid group, comprising the following steps:

[0007] (1) Mixing a recipient bacterial suspension with a sample solution containing donor bacteria from an environmental source, inoculating the mixture onto a sterile microporous filter membrane in a non-resistant solid culture medium, and performing a conjugation reaction;

[0008] (2) transferring the colonies on the sterile filter membrane in step (1) to a selection medium containing ampicillin, kanamycin and tetracycline for screening, and screening out colonies with fluorescent signals;

[0009] (3) extracting the DNA of the colonies obtained in step (2) for library construction, and loading it into the nanopore sequencing platform for selective sequencing using a real-time pop-up mode;

[0010] (4) removing the barcode and the adapter from the data obtained by selective sequencing in step (3), performing quality assessment, filtering out the data with an average quality score lower than 9, and performing de novo assembly on the filtered sequences to obtain assembled fragments;

[0011] (5) comparing the assembled fragments in step (4) with the genome of the recipient bacterium, and removing fragments with a similarity of ≥99%, a coverage of ≥90%, and no replication origin and mobile genetic elements;

[0012] (6) subjecting the assembled fragments in step (5) to one round of Medaka correction and three rounds of Racon correction;

[0013] Wherein, the recipient bacteria is Escherichia coli that simultaneously expresses fluorescent protein and carries kanamycin and tetracycline resistance genes; preferably, it is Escherichia coli gfp758.

[0014] In some embodiments, the ratio of the bacterial content of the recipient bacterial suspension in step (1) to the sample solution containing the donor bacteria from the environmental source is (3 to 10): 1. In the conjugation reaction, the contact frequency between the donor bacteria and the recipient bacteria directly affects the transfer efficiency of the plasmid. When the proportion of the number of recipient bacteria is high, more recipient cells are in a conjugable state. Once plasmid transfer occurs, the recipient strain with selection markers and fluorescence will be rapidly amplified, making it easier to screen out positive clones in the selection culture medium later and reduce background colony interference. The present application adjusts the ratio of the two to maximize the guarantee that the sequencing data is mainly based on the plasmid DNA obtained by conjugation, thereby improving the assembly success rate and the credibility of the results.

[0015] In some embodiments, the sample solution of the environmental source containing donor bacteria in step (1) includes any one of sludge, sewage, industrial wastewater, and surface water bodies. The effluent from sewage treatment plants, industrial emissions, and surface water are the main channels for the environmental transmission of resistance genes. The method of the present application has good environmental adaptability and universality, and is applicable to the enrichment and research of conjugative plasmids in real complex samples.

[0016] In some embodiments, the concentration of ampicillin in the selective medium in step (2) is 50 - 100 μg / mL, the concentration of kanamycin is 30 - 50 μg / mL, and the concentration of tetracycline is 10 - 20 μg / mL.

[0017] In some embodiments, the selective sequencing in step (3) includes: using the recipient bacterial genome sequence as the reference sequence for selective sequencing in the MinKNOW pop - up mode.

[0018] In some embodiments, the library construction step in step (3) includes:

[0019] (1) DNA extraction and purification;

[0020] (2) End - repairing the DNA;

[0021] (3) Ligating the corresponding barcodes and adapter sequences to the end - repaired DNA fragments.

[0022] In some embodiments, the objects of quality assessment in step (4) are N50, the number of sequences, the minimum length, and the maximum length.

[0023] In some embodiments, the fluorescent protein is any one of green fluorescent protein GFP, red fluorescent protein RFP, yellow fluorescent protein YFP, and blue fluorescent protein BFP.

[0024] In summary, compared with the prior art, the present invention has achieved the following technical effects:

[0025] 1. By combining the filter - mating method with selective sequencing, the present application successfully obtains a large number of transconjugants, has high efficiency in capturing conjugative plasmids in complex environmental samples, significantly improves the capture efficiency of conjugative plasmids, and has excellent capture and assembly capabilities for large - scale and highly complete plasmid genomes.

[0026] 2. Using the nanopore selective sequencing technology in the pop - up mode, the present application successfully realizes the pop - up exclusion of the E. coli gfp758 recipient genome, with a pop - up rate as high as 91.82%, significantly improving the purity of the conjugative plasmid sequence and the accuracy of subsequent assembly, and effectively excluding the interference of the recipient genome background.

[0027] 3. Compared with the traditional method, the α-diversity of the method of the present application has increased by 3.44 times, significantly enhancing the analytical depth of plasmid diversity in the microbial population, and the α-diversity of conjugative plasmids has increased significantly. In addition, up to 87.93% of the short fragment data can further expand the breadth and depth of the data on the basis of not losing the information of the traditional method by the method of the present application, with good data compatibility and coverage.

[0028] 4. The method of the present application is suitable for the plasmid research of complex environmental microbial communities, with high applicability and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is a flowchart of the method for efficiently capturing conjugative plasmidome of the present invention.

[0031] Figure 2 It is a comparison of the efficiency and coverage between the method of the present invention and the traditional method in plasmid capture; Figure a is a comparison of the coverage of long and short fragments for different-sized plasmids; Figure b is a comparison of the capture throughput of plasmid numbers by long and short reads; Figure c is a comparison of the proportion of plasmid fragments contained in long and short fragments. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0034] The present application efficiently captures the conjugative plasmidome by combining the filter membrane conjugation method and nanopore selective sequencing. The specific method is as follows:

[0035] 1. Filter membrane conjugation experiment:

[0036] (1) Preparation of recipient strains:

[0037] Strain selection: Escherichia coli sp.758 (E.coli gfp758) containing green fluorescent protein (GFP) and encoding kanamycin (KAN) and tetracycline (TET) resistance in its genome was selected as the recipient strain.

[0038] Cell preparation: The recipient cells were prepared by following the standard protocol for overnight culture in LB medium.

[0039] OD600 measurement: The OD600 value of the recipient strain was measured using a microplate spectrophotometer (Epoch2, BioTek Instruments).

[0040] Calculation of conversion factor: Based on the OD600 value and CFU value measured in the preliminary experiment, the conversion factor of the sample was calculated to facilitate the comparison of the numbers of environmental bacteria and recipient bacteria, and to facilitate the calculation of the donor-recipient ratio for subsequent experiments.

[0041] (2) Preparation of bacterial solution:

[0042] Dilution: The E.coli gfp758 solution and the cell suspension of each sludge sample were respectively diluted to an OD value of 0.047 (±0.003). Excessive bacterial numbers would affect the effect of conjugation.

[0043] Mixing: The recipient bacteria and donor bacteria were mixed at a quantity ratio of (3 - 10):1.

[0044] (3) Filter membrane conjugation reaction

[0045] The mixed bacterial solution was immediately dispensed onto a 25-mm diameter circular sterile filter membrane (GN-6 type, Pall Corporation, USA) in antibiotic-free LB medium and incubated overnight at 37°C.

[0046] (4) Plasmid screening and culture

[0047] On the next day, the filter membrane was inoculated onto a solid medium containing 100 μg / mL ampicillin (AMP), 50 μg / mL kanamycin (KAN), and 20 μg / mL tetracycline (TET). Subsequently, at least 1000 colonies showing fluorescence under green light excitation were picked using a sterile inoculation loop and inoculated into 5 mL of PBS solution. After vortex mixing the PBS solution, the culture was continued overnight at the same antibiotic concentration. The culture was collected for subsequent sequencing analysis.

[0048] AMP, as a resistance marker for plasmid capture, is a typical β-lactam antibiotic and is universal in screening for mobile plasmid-mediated microbial resistance. Therefore, this filter membrane conjugation experiment captured conjugation plasmids with AMP resistance.

[0049] 2. Selective sequencing

[0050] Selective sequencing effectively enriches plasmidome fragments by using the pop-up mode of MinKNOW to reject DNA fragments that match the genome of strain E. coli gfp758 from entering.

[0051] The specific steps are as follows:

[0052] (1) DNA extraction and library construction

[0053] Total DNA was extracted from the green fluorescent bacteria, and a library was constructed using the SQK-LSK109 kit (V14, Oxford Nanopore Technologies Ltd., UK). The DNA was purified using AMPure XP magnetic beads (Beckman Coulter, Inc., USA). End repair was performed on high molecular weight DNA of more than 400 ng, and the reagents used included FFPE DNA Repair Buffer, FFPE DNA Repair Mix, Ultra II End-prep Reaction Buffer, and Ultra II End-prep Enzyme Mix (NEB, UK). Subsequently, the repaired DNA fragments were further processed to ligate the corresponding barcodes and adapter sequences for downstream applications.

[0054] (2) Sequencing preparation and MinKNOW pop-up mode setting

[0055] The constructed library was loaded into a SpotON Flow Cell (R9.4.1) FLO-MIN106 flow cell and then inserted into a GridION X5 instrument for selective sequencing.

[0056] Using the pop-up mode of MinKNOW (version 21.10.5), the complete genome of E. coli gfp758 (1 circular fragment, size 4.36 Mb, average coverage approximately 100×) was set as the reference sequence. Adaptive sequencing parameters in Deplete mode were used for selective sequencing to enrich plasmidome fragments.

[0057] (3) Data analysis and processing

[0058] For nanopore selective sequencing data, the initial steps included using Porechop to remove barcodes and adapters; using NanoFilt to screen out low-quality data with an average quality score below 9. NanoPlot was used for quality assessment, including evaluating metrics such as N50, number of sequences, minimum length, and maximum length; metaFlye was used to perform de novo assembly on the screened clean sequences to recover the plasmid genome.

[0059] Since nanopore selective sequencing failed to completely exclude the E. coli gfp758 genomic sequence, bacterial fragments need to be removed after assembling all selective sequencing fragments. Specifically: By using the LAST tool to globally align the assembled fragments with the E. coli gfp758 genome, fragments with a similarity greater than or equal to 99% and a coverage greater than or equal to 90% are deleted. In addition, it is also necessary to ensure that the finally assembled fragments have plasmid characteristics, such as the origin of replication (ori) and mobile elements. Any fragments that do not conform to these characteristics will be discarded; Use long-read sequence data for one round of Medaka correction and three rounds of Racon correction to further improve the quality of plasmid fragments.

[0060] Application Example 1

[0061] As Figure 1 shown, the biological treatment system (influent, anaerobic sludge, anoxic sludge, and aerobic sludge) of Shenzhen Gucheng Sewage Treatment Plant was selected as the sampling point, and the filter membrane conjugation method was used to conduct a plasmid conjugation transfer experiment with Escherichia coli containing green fluorescence as the recipient and the above samples as the donor at a mixing ratio of 5:1.

[0062] Positive colonies were screened according to their ampicillin, kanamycin, and tetracycline resistance and green fluorescence, and 2123 (influent), 2141 (anaerobic sludge), 2303 (anoxic sludge), and 2084 (aerobic sludge) transconjugants were obtained respectively.

[0063] The total genomic DNA of the positive clones obtained through the filter membrane conjugation experiment was sequenced using nanopore selective sequencing in the pop-up mode, with the goal of excluding the E. coli gfp758 genome. In the selective sequencing experiment, the pop-up rate of the E. coli gfp758 genome was 91.82%. After self-correction of the third-generation data for the above nanopore data, a total of 1.1 million plasmid fragments with a maximum length of 65.82 kb were obtained, and all the data were used for de novo assembly of the plasmidome. Finally, 622 non-redundant plasmid genomes were assembled, with an average length of 24.21 kb and a maximum length of 202.81 kb.

[0064] Application Example 2

[0065] In addition, the method for efficiently capturing the conjugative plasmidome of this application was compared with the plasmidome obtained by the traditional capture method based on a plasmid extraction kit (Qiagen Plasmid Kit) and through Illumina metagenomic sequencing.

[0066] The results are as Figure 2As shown in a and b, the plasmid groups captured by this method were compared with those obtained by Illumina sequencing using a plasmid extraction kit. Among the 622 non-redundant plasmids, as many as 441 conjugative plasmids were only captured by the method of this application and not obtained from traditional capture methods. 181 plasmids were captured for both short and long fragments. This indicates that the α-diversity of plasmids captured by the method of this application is 3.44 times higher than that of traditional methods. In addition, as Figure 2 shown in c, 87.93% of the short fragments could be aligned to the plasmids assembled by nanopore selective sequencing from the extracted plasmid group dataset, which also fully demonstrates that the plasmid group sequences captured by the method of this application basically cover the plasmid genomes captured by traditional methods.

[0067] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An efficient method for capturing conjugative plasmidomes, characterized in that, The steps include: (1) Mixing a recipient bacterial suspension with a sample solution containing donor bacteria from an environmental source, inoculating the mixture onto a sterile microporous filter membrane in a non-resistant solid culture medium, and performing a conjugation reaction; (2) transferring the colonies on the sterile filter membrane in step (1) to a selection medium containing ampicillin, kanamycin and tetracycline for screening, and selecting the conjugant colonies with fluorescent signals; (3) extracting DNA from the conjugant colonies obtained in step (2) for library construction, and loading it onto a nanopore sequencing platform for selective sequencing using a real-time pop-up mode; (4) removing the barcode and the adapter from the data obtained by selective sequencing in step (3), performing quality assessment, filtering out the data with an average quality score lower than 9, and performing de novo assembly on the filtered sequences to obtain assembled fragments; (5) comparing the assembled fragments in step (4) with the genome of the recipient bacterium, and removing fragments with a similarity of ≥99%, a coverage of ≥90%, and no replication origin and mobile genetic elements; (6) subjecting the assembled fragments in step (5) to one round of Medaka correction and three rounds of Racon correction; The recipient bacteria are Escherichia coli that simultaneously express fluorescent protein and carry kanamycin and tetracycline resistance genes.

2. The method according to claim 1, wherein The ratio of the bacterial content of the recipient bacterial suspension in step (1) to the bacterial content of the sample solution containing donor bacteria from the environmental source is (3-10):

1.

3. The method according to claim 1, characterized in that, The sample solution containing donor bacteria from the environmental source in step (1) includes any one of sludge, sewage, industrial wastewater and surface water.

4. The method according to claim 1, wherein In step (2), the concentration of ampicillin in the selection medium is 50-100 μg / mL, the concentration of kanamycin is 30-50 μg / mL, and the concentration of tetracycline is 10-20 μg / mL.

5. The method according to claim 1, wherein The selective sequencing in step (3) includes: using the recipient bacterial genome sequence as a reference sequence for selective sequencing in the MinKNOW pop-up mode.

6. The method according to claim 1, characterized in that, The library construction step in step (3) includes: (1) DNA extraction and purification; (2) Perform end repair on DNA; (3) Connect the corresponding barcode and adapter sequences to the end-repaired DNA fragments.

7. The method according to claim 1, wherein The objects of quality assessment in step (4) are N50, number of sequences, minimum length and maximum length.

8. The method according to claim 1, wherein The fluorescent protein is any one of green fluorescent protein GFP, red fluorescent protein RFP, yellow fluorescent protein YFP and blue fluorescent protein BFP.

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