CN113782100A - Method for identifying plasmid type carried by bacterial population based on bacterial genome high-throughput sequencing data - Google Patents
Method for identifying plasmid type carried by bacterial population based on bacterial genome high-throughput sequencing data Download PDFInfo
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Abstract
The invention discloses a method for identifying types of plasmids carried by a bacterial population based on bacterial genome high-throughput sequencing data. The method is used for identifying and typing plasmids contained in the bacteria by analyzing and splicing high-throughput sequencing data of a plurality of bacterial strains. The invention can analyze a large amount of bacteria sequencing data in a short time to obtain the plasmid types, drug resistance/virulence genes and the like contained in the bacteria sequencing data. The invention can be used as a beneficial supplement of the traditional laboratory identification biological experiment, and achieves the purpose of quickly identifying the plasmid. The invention has important application value.
Description
Technical Field
The invention belongs to bioinformatics, and particularly relates to a method for identifying types of plasmids carried by a bacterial population based on bacterial genome high-throughput sequencing data, in particular to a method for analyzing and splicing all high-throughput sequencing data of bacteria by combining plasmid sequences and types found by existing target bacteria, and identifying and typing plasmids contained in the bacteria.
Background
A plasmid is a DNA molecule capable of autonomous replication outside the chromosomal or nuclear region DNA of a cell. One or more plasmid types are present in a bacterial cell. The plasmid is an important functional element in bacteria, and can carry drug-resistant or virulence genes (such as plasmids pXO1 and pXO2 which play a decisive role in the virulence of bacillus anthracis and tetracycline-resistant genes contained in escherichia coli). The plasmid can be transferred among different bacterial strains, and in the process, the horizontal transfer of drug-resistant and virulence genes in the bacteria can be realized, so that the cells are endowed with additional physiological metabolic capacity, and the pathogenicity of the cells is improved. The type and the quantity of the plasmids in the bacterial population are clarified, and the plasmid has important functions on researching the drug resistance and the virulence characteristics of the bacteria, preventing and controlling bacterial infection and further transmitting the bacterial infection.
High throughput sequencing technologies can obtain large amounts of bacterial genomic data in a short time, which provides conditions and challenges for systematic study of plasmid types and their distribution in bacterial populations. How to identify the types, characteristics and distribution conditions of plasmids carried by bacterial populations from massive bacterial genome high-throughput sequencing data is a problem to be solved urgently.
Disclosure of Invention
The invention aims to identify the type of plasmid carried by a bacterial population and whether a drug resistance gene and/or a virulence gene is located on the plasmid.
The invention firstly protects a method for identifying the plasmid type carried by a bacterial population and whether a drug-resistant gene and/or a virulence gene are/is located on a plasmid based on bacterial genome high-throughput sequencing data, which comprises the following steps:
(c1) splicing genome high-throughput sequencing data of different strains of bacteria respectively to obtain splicing sequences of the strains;
(c2) after the step (c 1) is completed, comparing the splicing sequences of different strains with the plasmid database respectively to obtain the splicing sequences containing the plasmid replicon; obtaining a plasmid type according to the plasmid replicon, namely the plasmid type carried by the bacterial population;
(c3) after the step (c 1) is finished, the splicing sequences of different strains are respectively compared with a drug resistance gene database and/or a virulence gene database to obtain splicing sequences containing drug resistance genes and/or virulence genes;
(c4) dividing the reads coverage depth of the plasmid replicon by the reads coverage depth of the drug resistance gene and/or the virulence gene to obtain a reads coverage depth ratio; then, the following judgment is made: if the ratio of the coverage depth of reads is above 0.90 and below 1.10, the drug resistance gene and/or virulence gene is on the plasmid; otherwise, the drug resistance gene and/or virulence gene is not on the plasmid;
the method is useful for diagnosis and treatment of non-diseases.
The invention also provides a method for identifying the type of plasmid carried by a bacterial population based on the bacterial genome high-throughput sequencing data, which comprises the following steps:
(a1) splicing genome high-throughput sequencing data of different strains of bacteria respectively to obtain splicing sequences of the strains;
(a2) after the step (a 1) is completed, comparing the splicing sequences of different strains with a plasmid database respectively, and obtaining plasmid types, namely the plasmid types carried by the bacterial population, according to the plasmid replicons;
the method is useful for diagnosis and treatment of non-diseases.
The invention also provides a method for identifying whether a drug-resistant gene and/or a virulence gene in a bacterial population is located on a plasmid based on the bacterial genome high-throughput sequencing data, which comprises the following steps:
(b1) splicing genome high-throughput sequencing data of different strains of bacteria respectively to obtain splicing sequences of the strains;
(b2) after the step (b 1) is completed, comparing the splicing sequences of different strains with the plasmid database respectively to obtain the splicing sequences containing the plasmid replicon;
(b3) after the step (b 1) is finished, the splicing sequences of different strains are respectively compared with a drug resistance gene database and/or a virulence gene database to obtain splicing sequences containing drug resistance genes and/or virulence genes;
(b4) dividing the reads coverage depth of the plasmid replicon by the reads coverage depth of the drug resistance gene and/or the virulence gene to obtain a reads coverage depth ratio; then, the following judgment is made: if the ratio of the coverage depth of reads is above 0.90 and below 1.10, the drug resistance gene and/or virulence gene is on the plasmid; otherwise, the drug resistance gene and/or virulence gene is not on the plasmid;
the method is useful for diagnosis and treatment of non-diseases.
The invention also provides the use of any of the methods described above for identifying whether a bacterium contains a plasmid or the type of plasmid that the bacterium contains;
the use is for the diagnosis and treatment of non-diseases.
The invention also protects the application of any one of the methods in identifying the drug resistance and/or virulence of the bacteria;
the use is for the diagnosis and treatment of non-diseases.
In the above applications, the bacterial genome contains a drug resistance gene, i.e., the bacteria have drug resistance.
In the application, the bacterial genome contains virulence genes, namely the bacteria have corresponding virulence.
The invention also protects the application of any one of the methods in identifying whether the bacterial plasmid contains a drug resistance gene and/or virulence;
the use is for the diagnosis and treatment of non-diseases.
Any of the above bacteria may be providencia.
Any one of the plasmid databases described above may be the plasmid database Plasmidfinder.
Any one of the above-mentioned drug-resistant gene databases may be a drug-resistant gene database Resfinder.
Any of the virulence gene databases described above may be the virulence gene database VFDB.
Any one of the resistance genes is blaNDM and/or blaIMP.
Any one of the above plasmid types is IncC, IncpGZH766-NDM, IncFIip911012-NDM, IncpPrY2001 or IncW.
In one embodiment of the present invention, 257 providencia strains were detected by the above method. The results show that 257 providencia contains plasmids including five plasmid types including IncC, IncpGZH766-NDM, IncFIip911012-NDM, IncpPrY2001 and IncW, and the plasmids contain two drug resistance genes, namely blaNDM and blaIMP.
The invention establishes a method for identifying the plasmid type carried by a bacterial population based on bacterial genome high-throughput sequencing data, and can analyze a large amount of bacterial sequencing data in a short time to obtain the plasmid type, drug resistance/virulence genes and the like contained in the bacterial sequencing data. The invention can be used as a beneficial supplement of the traditional laboratory identification biological experiment, and achieves the purpose of quickly identifying the plasmid. The invention has important application value.
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FIG. 1 is a schematic diagram of a process for identifying the types of plasmids carried by a bacterial population and whether a target gene is located on the plasmids based on high-throughput sequencing data of bacterial genomes.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 establishment of a method for identifying the type of plasmid carried by a bacterial population and whether a target gene is located on the plasmid based on bacterial genome high-throughput sequencing data
Through a large number of experiments, the inventor of the invention focuses on high-throughput sequencing data of bacterial genomes, combines existing plasmid sequence resources of a public database, and adopts a bioinformatics means to establish a method for identifying the plasmid type carried by the bacterial population and whether a target gene (a virulence gene and/or a drug resistance gene) is positioned on a plasmid based on the high-throughput sequencing data of the bacterial genomes. The method comprises the following specific steps:
1. and splicing the bacterial genome high-throughput sequencing data to obtain a bacterial splicing sequence.
2. After the step 1 is completed, comparing the splicing sequence of the bacteria with a plasmid database (comprising a public database and a database collected and arranged in a laboratory) to obtain a splicing sequence containing a plasmid replicon; and analyzing the splicing sequence containing the plasmid replicon to obtain the plasmid type contained in the splicing sequence of the bacteria, namely the plasmid type carried by the bacterial population.
3. After the step 1 is completed, the splicing sequence of the bacteria is respectively compared with a drug resistance gene database and a virulence gene database (comprising a public database and a database collected and arranged in a laboratory) to obtain the splicing sequence containing the drug resistance gene and/or the virulence gene; and (3) positioning the sequence of the drug resistance gene and/or the virulence gene.
4. Dividing the reads coverage depth of the plasmid replicon by the reads coverage depth of the drug resistance gene and/or the virulence gene to obtain the reads coverage depth ratio. Then, the following judgment is made: if the ratio of the coverage depth of reads is above 0.90 and below 1.10, the drug resistance gene and/or virulence gene is on the plasmid; otherwise the drug resistance gene and/or virulence gene are not on the plasmid.
The schematic flow chart of the method for identifying the plasmid type carried by the bacterial population and whether the target gene is located on the plasmid based on the bacterial genome high-throughput sequencing data is shown in figure 1.
Example 2 application of the method established in example 1
Research has shown that providencia forms 7 complex groups, namely S01-S07. The present inventors obtained 257 providencia strains from 36 hospitals from 12 municipalities of provincial municipalities in china.
The basic information (including strain name, sampling site, year, age, sex, specimen, disease and department) of 257 providencia is shown in Table 1.
1. Obtaining of splicing sequence of providencia
(1) Providencia is subjected to high-throughput sequencing and then filtered (for quality control) to obtain high-quality sequencing data, namely providencia genome high-throughput sequencing data.
And detecting 257 providencia sp, and obtaining genome high-throughput sequencing data of 257 providencia sp.
(2) And splicing the genome high-throughput sequencing data of 257 providencia strains by using Spades software to obtain spliced sequences of 257 providencia strains.
2. After the step 1 is completed, taking the data of the splicing sequence of 257 providencia as query, searching plasmid database plasmid finder, and obtaining the plasmid type according to rep gene (plasmid replicon); and recording the contig where the rep gene is located (denoted as contig _ rep).
3. After the step 1 is completed, using the data of the splicing sequence of 257 providencia as query, searching a drug-resistant gene database Resfinder and a virulence gene database VFDB, and recording the contig (marked as contig _ gene) of the drug-resistant gene and/or the virulence gene.
4. Obtaining reads coverage depth of contig _ rep according to formula 1; contig _ readheadsdepth= num (reads) × len (reads)/len (contig _ rep) (formula 1);
wherein contig _ readheadsdepth"reads coverage depth for contig _ rep", "num (reads) is the number of reads, len (reads) is the average length of reads sequenced, and len (contig _ rep) is the length of contig where the rep gene is located.
5. Obtaining the reads coverage depth of the contig _ gene according to a formula 2; contig _ genesdepth= num (reads) × len (reads)/len (containment _ gene) (formula 2);
among them, contig _ genesdepthFor the depth of coverage of reads for contig _ gene, num (reads) is the number of reads, len (reads) is the average length of reads sequenced, len (contig _ gene) is the length of contig where the genes other than rep are located.
6. After the steps 4 and 5 are completed, dividing the reads coverage depth of the contig _ rep by the reads coverage depth of the contig _ gene to obtain a reads coverage depth ratio; the following judgment is made: if the ratio of the coverage depth of reads is above 0.90 and below 1.10, the drug resistance gene and/or virulence gene is on the plasmid; otherwise the drug resistance gene and/or virulence gene are not on the plasmid.
The results are shown in Table 2. The results showed that 257 providencia strains were present in 5 complex groups; 257 providencia strains contain plasmids comprising five plasmid types including IncC, IncpGZH766-NDM, IncFIip911012-NDM, IncpPrY2001 and IncW, and the plasmids contain two drug resistance genes which are blaNDM and blaIMP respectively; most of the plasmids were localized to providencia SP01, and the IncC-type plasmid was widely present in all providencia.
The conventional method for detecting the plasmid type and the drug-resistant gene contained in the bacteria needs to carry out third-generation sequencing to obtain a cyclized plasmid complete sequence, and then, the plasmid type and the drug-resistant gene are judged to be searched and compared. This process is not only cumbersome, but also time consuming and expensive. The method is based on cheaper second-generation sequencing data, does not need to obtain the full sequence of the plasmid, can judge the type of the plasmid and the drug-resistant gene contained in the plasmid through the reads characteristic, is simple and convenient, is easy to operate and is more economic. Therefore, the method provided by the invention has important application value.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A method for identifying the plasmid type carried by a bacterial population and whether a drug-resistant gene and/or a virulence gene are located on a plasmid based on bacterial genome high-throughput sequencing data, comprising the following steps:
(c1) splicing genome high-throughput sequencing data of different strains of bacteria respectively to obtain splicing sequences of the strains;
(c2) after the step (c 1) is completed, comparing the splicing sequences of different strains with the plasmid database respectively to obtain the splicing sequences containing the plasmid replicon; obtaining a plasmid type according to the plasmid replicon, namely the plasmid type carried by the bacterial population;
(c3) after the step (c 1) is finished, the splicing sequences of different strains are respectively compared with a drug resistance gene database and/or a virulence gene database to obtain splicing sequences containing drug resistance genes and/or virulence genes;
(c4) dividing the reads coverage depth of the plasmid replicon by the reads coverage depth of the drug resistance gene and/or the virulence gene to obtain a reads coverage depth ratio; then, the following judgment is made: if the ratio of the coverage depth of reads is above 0.90 and below 1.10, the drug resistance gene and/or virulence gene is on the plasmid; otherwise, the drug resistance gene and/or virulence gene is not on the plasmid;
the method is useful for diagnosis and treatment of non-diseases.
2. A method for identifying the type of plasmid carried by a bacterial population based on high throughput sequencing data of bacterial genomes, comprising the following steps:
(a1) splicing genome high-throughput sequencing data of different strains of bacteria respectively to obtain splicing sequences of the strains;
(a2) after the step (a 1) is completed, comparing the splicing sequences of different strains with a plasmid database respectively, and obtaining plasmid types, namely the plasmid types carried by the bacterial population, according to the plasmid replicons;
the method is useful for diagnosis and treatment of non-diseases.
3. A method for identifying whether a drug-resistant gene and/or a virulence gene in a bacterial population is located on a plasmid based on high throughput sequencing data of bacterial genomes, comprising the steps of:
(b1) splicing genome high-throughput sequencing data of different strains of bacteria respectively to obtain splicing sequences of the strains;
(b2) after the step (b 1) is completed, comparing the splicing sequences of different strains with the plasmid database respectively to obtain the splicing sequences containing the plasmid replicon;
(b3) after the step (b 1) is finished, the splicing sequences of different strains are respectively compared with a drug resistance gene database and/or a virulence gene database to obtain splicing sequences containing drug resistance genes and/or virulence genes;
(b4) dividing the reads coverage depth of the plasmid replicon by the reads coverage depth of the drug resistance gene and/or the virulence gene to obtain a reads coverage depth ratio; then, the following judgment is made: if the ratio of the coverage depth of reads is above 0.90 and below 1.10, the drug resistance gene and/or virulence gene is on the plasmid; otherwise, the drug resistance gene and/or virulence gene is not on the plasmid;
the method is useful for diagnosis and treatment of non-diseases.
4. A method according to any of claims 1 to 3, characterized by: the bacteria are providencia.
5. A method according to any of claims 1 to 3, characterized by: the plasmid database is a plasmid database Plasmidfinder.
6. A method according to claim 1 or 3, characterized by: the drug-resistant gene database is a drug-resistant gene database Resfinder.
7. A method according to claim 1 or 3, characterized by: the virulence gene database is a virulence gene database VFDB.
8. Use of the method of any one of claims 1 to 3 for identifying whether a bacterium contains a plasmid or a type of plasmid contained by a bacterium;
the use is for the diagnosis and treatment of non-diseases.
9. Use of the method of any one of claims 1 to 3 for identifying bacterial resistance and/or virulence;
the use is for the diagnosis and treatment of non-diseases.
10. Use of the method of any one of claims 1 to 3 to identify whether a bacterial plasmid contains a drug resistance gene and/or virulence;
the use is for the diagnosis and treatment of non-diseases.
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