Method for efficiently recognizing intracellular mobile genetic elements of bacterial plasmids

Abstract

The invention discloses a method for efficiently recognizing intracellular mobile genetic elements of bacterial plasmids, and belongs to the field of microbial bioinformatics analysis. Comprising the following steps: 1, establishing a database by using a genome sequence of a to-be-analyzed plasmid, and searching the genome sequence of the to-be-analyzed plasmid in the database by using a gene sequence of a mobile genetic element in a bacterial plasmid cell as a query sequence; 2, screening the search results; identifying the screening result according to an identification standard to obtain an independent identification result of the intracellular mobile genetic element type contained in the to-be-analyzed plasmid; and 3, combining the recognition results of the intracellular mobile genetic elements of the plasmids to be analyzed, and obtaining the final recognition result of the intracellular mobile genetic elements of the plasmids to be analyzed according to the recognition standard. The method provided by the invention has the beneficial effects that automatic batch processing can be realized, and the intracellular mobile genetic elements of the bacterial plasmids can be efficiently, accurately and conveniently identified.

Description

Technical Field

[0001] The present application relates to the field of microbial bioinformatics analysis, and in particular, to a method for efficiently identifying mobile genetic elements within bacterial plasmids. Background Art

[0002] There are a variety of intracellular mobile genetic elements in bacterial plasmid genomes. Intracellular mobile genetic elements refer to DNA fragments that can move within the genome, usually including transposons, integrons, and insertion sequences. These intracellular mobile genetic elements have attracted much attention because they often contain a variety of drug resistance genes and virulence factors, have the characteristics of moving within the genome, and can mediate the widespread dissemination of drug resistance genes. Therefore, identifying the distribution area and type of mobile genetic elements from bacterial plasmid genome sequences is of great significance for plasmid genome analysis.

[0003] The current method of identifying mobile genetic elements in bacterial plasmid genomes usually uses online tools or localized tools. The method of using online tools for identification is to submit the genome sequence of bacterial plasmids on the website and wait for the results to be returned.

[0004] This solution has the following disadvantages:

[0005] 1. This method is only applicable to the search of mobile genetic elements in a small number of plasmid genome sequences. Since only one genome sequence can be uploaded at a time, it is difficult to efficiently and quickly complete the task of identifying mobile genetic elements when faced with a large number of microbial genome sequences.

[0006] 2. The recognition speed of mobile genetic elements is slow. The recognition speed of mobile genetic elements based on the web version is limited by the number of real-time visitors to the website and the network speed, so the recognition speed is usually slow.

[0007] The method of using localization tools for identification is to perform local homology search and comparison based on the existing integron, transposon and insertion sequence data. This method also has certain disadvantages:

[0008] This method can only search for integrons, transposons, and insertion sequences separately, resulting in a high false positive rate for the identified mobile genetic elements. In bacterial plasmid genomes, mobile genetic elements are often nested. For example, a transposon contains multiple insertion sequences and integrons. When searching for intracellular mobile genetic elements separately, it is easy to repeatedly identify the same gene region, making it difficult to accurately determine the specific type and distribution characteristics of intracellular mobile genetic elements.

[0009] Therefore, there is a need for a method that can realize automated batch processing, is efficient, accurate, and convenient for efficiently identifying mobile genetic elements in bacterial plasmids. Summary of the invention

[0010] The content of this application is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this application is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[0011] In order to solve the technical problems mentioned in the above background technology section, some embodiments of the present application provide a method for efficiently identifying mobile genetic elements in bacterial plasmids, comprising the following steps:

[0012] Step 1: Establish a database with the genome sequence of the plasmid to be analyzed, use the gene sequence of the mobile genetic element in the bacterial plasmid as the query sequence, and search the genome sequence of the plasmid to be analyzed in the database;

[0013] Step 2: Screening the search results; identifying the screening results according to the identification criteria to obtain a separate identification result of the type of intracellular mobile genetic element contained in the plasmid to be analyzed;

[0014] Step 3: Merge the identification results of each plasmid intracellular mobile genetic element to be analyzed, and obtain the final identification result of the plasmid intracellular mobile genetic element to be analyzed according to the identification criteria.

[0015] Furthermore, the intracellular mobile genetic elements include transposons, integrons and insertion sequences.

[0016] Furthermore, in step 1, the gene sequence of the bacterial plasmid intracellular mobile genetic element is collected and obtained through public literature and public databases.

[0017] Furthermore, in step 1, the search method is: running a local Blastn tool in a computer system, using the gene sequences of the transposon, integron and insertion sequence as query sequences, and performing a Blastn search on the gene sequence of the plasmid to be analyzed.

[0018] Furthermore, in step 2, the screening method is: screening the search results according to the conventional standards for homologous sequence comparison.

[0019] Furthermore, the conventional standards for homologous sequence alignment are: e-value less than 0.001, identity greater than or equal to 50% and coverage greater than or equal to 50%.

[0020] Furthermore, the identification criteria are:

[0021] In a single plasmid, when there is a complete containment relationship between two intracellular mobile genetic elements, the recognition results with a wider range of intervals are retained;

[0022] When there is a crossover region between two intracellular mobile genetic elements, and the crossover region is less than or equal to 50 bp, it is judged as two different intracellular mobile genetic element identification results;

[0023] When there is a crossover region between two intracellular mobile genetic elements, and the crossover region is greater than 50 bp, the identification result with the longer length is retained;

[0024] When there is an intersection region between two intracellular mobile genetic elements and the non-intersection region is less than 50 bp, they are judged as the same recognition result, and the recognition result with a longer length is retained.

[0025] The beneficial effect of the present application is that it provides a method for efficiently identifying mobile genetic elements in bacterial plasmids that can realize automated batch processing and is efficient, accurate and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings constituting a part of this application are used to provide a further understanding of this application, so that other features, purposes and advantages of this application become more obvious. The illustrative embodiment drawings and their descriptions of this application are used to explain this application and do not constitute an improper limitation on this application.

[0027] In addition, throughout the drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and that the components and elements are not necessarily drawn to scale.

[0028] In the attached picture:

[0029] Figure 1 It is a schematic diagram of the overall process according to an embodiment of the present application;

[0030] Figure 2 It is a possible mode of recognition of mobile genetic elements in bacterial plasmid genomes. DETAILED DESCRIPTION

[0031] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0032] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0033] It should be noted that the concepts such as "first" and "second" mentioned in the present disclosure are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence of the functions performed by these devices, modules or units.

[0034] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0035] The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0036] Embodiment, a method for efficiently identifying mobile genetic elements in bacterial plasmid cells, referring to Figure 1 , including the following steps:

[0037] Step 1: Establish a database with the genome sequence of the plasmid to be analyzed, use the gene sequence of the mobile genetic element in the bacterial plasmid as the query sequence, and search the genome sequence of the plasmid to be analyzed in the database; the gene sequence of the mobile genetic element in the bacterial plasmid is collected and obtained through public literature and public databases; the search method is: run the local Blastn tool in the computer system, use the gene sequences of the transposon, integron and insertion sequence as the query sequence, and perform Blastn search on the gene sequence of the plasmid to be analyzed; the specific process is as follows:

[0038] Create a working folder Plasmid_analysis and a subfolder Plasmids. Download the plasmid nucleic acid genome sequences with accession numbers NZ_CP017387 and NZ_AP022395 from the public database, merge the two genome sequences into one file, name it Plasmid_sequences.fasta, and save it in the Plasmids folder.

[0039] Run the local Blastn tool, use the Plasmid_sequences.fasta sequence to create a database named Plasmid_sequences_database, use the insertion sequence / integrons / transposons sequences Insertion.fasta / Integron.fasta / Transposon.fasta as queries, perform Blastn searches, and name the output results Insertion_result.txt, Integron_result.txt, and Transposon_result.txt;

[0040] The instruction to create a database is:

[0041] "makeblastdb-in Plasmid_sequences.fasta-dbtype nucl-parse_seqids-hash_index-out Database\Plasmid_sequences_database";

[0042] The command for Blastn search is:

[0043] "blastn-query Insertion.fasta\Integron.fasta\Transposon.fasta-dbDatabase\Plasmid_sequences_database-outfmt" 6qseqid sseqid pident length qcovsqcovhsp qcovus mismatch gapopen qstart qend sstart send evalue bitscore"-outInsertion_result.txt\Integron_result.txt\Transposon_result.txt".

[0044] Step 2: Screen the search results using conventional standards for homologous sequence alignment; identify the screening results according to the identification criteria to obtain a separate identification result of the type of intracellular mobile genetic element contained in the plasmid to be analyzed;

[0045] The conventional standards for homologous sequence alignment are: e-value less than 0.001, identity greater than or equal to 50% and coverage greater than or equal to 50%; based on this standard, the search results of transposons, integrons and insertion sequences of the plasmid to be analyzed are screened in turn. Based on the collected classification information of bacterial plasmid transposons, integrons and insertion sequences, the results obtained by the Blastn search are annotated with types. During the screening process, if a sequence is aligned to different intracellular mobile genetic elements multiple times, only the result with the largest identity is retained; when the identity is the same, the result with a larger coverage is retained; when both the identity and coverage are the same, only the result with a smaller e-value is retained;

[0046] The identification criteria are:

[0047] In a single plasmid, when there is a complete containment relationship between two intracellular mobile genetic elements, the recognition results with a wider range of intervals are retained;

[0048] When there is no complete inclusion relationship between two intracellular mobile genetic elements, but there is an intersection region, and the intersection region is less than or equal to 50 bp, it is judged as two different intracellular mobile genetic element identification results;

[0049] When there is no complete inclusion relationship between two intracellular mobile genetic elements, but there is a crossover region, and the crossover region is greater than 50 bp, the identification result with a longer length is retained;

[0050] When there is a large fragment intersection region between two intracellular mobile genetic elements, and the non-intersection region is less than 50 bp, they are judged as the same recognition result, and the recognition result with a longer length is retained;

[0051] The recognition results obtained by the above recognition criteria are:

[0052] Insertion sequence of NZ_AP022395:

[0053]

[0054] Integron of NZ_AP022395:

[0055]

[0056] Transposon of NZ_AP022395:

[0057]

[0058]

[0059] Insertion sequence of NZ_CP030132:

[0060]

[0061]

[0062] Integron of NZ_CP030132:

[0063]

[0064]

[0065] Transposon of NZ_CP030132:

[0066]

[0067]

[0068] Step 3: Merge the insertion sequence, integron, and transposon recognition results of each plasmid to be analyzed, and obtain the final recognition result of the intracellular mobile genetic element of the plasmid to be analyzed according to the recognition criteria;

[0069] The insertion sequence, integron and transposon recognition results of NZ_AP022395 and NZ_CP030132 plasmids were combined, and the final recognition results were obtained according to the recognition criteria:

[0070] The intracellular mobile genetic elements contained in NZ_AP022395 are:

[0071]

[0072]

[0073] The intracellular mobile genetic elements contained in NZ_CP030132 are:

[0074]

[0075]

[0076] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

[0077] In summary, the method of the present application can improve the accuracy and convenience of identifying mobile genetic elements in bacterial plasmids. Based on data downloaded from public literature and public databases, it can realize automated and batch processing to achieve efficient identification of mobile genetic elements in bacterial plasmids.

Claims

1. A method for efficiently identifying mobile genetic elements in bacterial plasmids, characterized in that: The following steps are involved: Step 1: Establish a database with the genome sequence of the plasmid to be analyzed, use the gene sequence of the mobile genetic element in the bacterial plasmid as the query sequence, and search the genome sequence of the plasmid to be analyzed in the database; Step 2: Screening the search results; identifying the screening results according to the identification criteria to obtain a separate identification result of the type of intracellular mobile genetic element contained in the plasmid to be analyzed; Step 3: Merge the identification results of each plasmid intracellular mobile genetic element to be analyzed, and obtain the final identification result of the plasmid intracellular mobile genetic element to be analyzed according to the identification criteria.

2. The method for efficiently identifying mobile genetic elements in bacterial plasmids according to claim 1, characterized in that: The intracellular mobile genetic elements include transposons, integrons and insertion sequences.

3. The method for efficiently identifying mobile genetic elements in bacterial plasmids according to claim 2, characterized in that: In step 1, the gene sequence of the bacterial plasmid intracellular mobile genetic element is collected and obtained through public literature and public databases.

4. The method for efficiently identifying mobile genetic elements in bacterial plasmids according to claim 3, characterized in that: In step 1, the search method is: running a local Blastn tool in a computer system, using the gene sequences of the transposon, integron and insertion sequence as query sequences, and performing a Blastn search on the gene sequence of the plasmid to be analyzed.

5. The method for efficiently identifying mobile genetic elements in bacterial plasmids according to claim 4, characterized in that: In step 2, the screening method is: screening the search results according to the conventional standards for homologous sequence comparison.

6. The method for efficiently identifying mobile genetic elements in bacterial plasmids according to claim 5, characterized in that: The conventional standards for homologous sequence alignment are: e-value less than 0.001, identity greater than or equal to 50% and coverage greater than or equal to 50%.

7. The method for efficiently identifying mobile genetic elements in bacterial plasmids according to claim 6, characterized in that: The identification criteria are: In a single plasmid, when there is a complete containment relationship between two intracellular mobile genetic elements, the recognition results with a wider range of intervals are retained; When there is a crossover region between two intracellular mobile genetic elements, and the crossover region is less than or equal to 50 bp, it is judged as two different intracellular mobile genetic element identification results; When there is a crossover region between two intracellular mobile genetic elements, and the crossover region is greater than 50 bp, the identification result with the longer length is retained; When there is an intersection region between two intracellular mobile genetic elements and the non-intersection region is less than 50 bp, they are judged as the same recognition result, and the recognition result with a longer length is retained.

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