CN112309499A - Method and device for quickly annotating bacterial pdif - Google Patents
Method and device for quickly annotating bacterial pdif Download PDFInfo
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Abstract
The invention discloses a method and a device for quickly annotating bacterial pdif, which are used for constructing a pdifDB database; obtaining a bacterial DNA sequence to be analyzed through whole genome sequencing; the bacterial DNA sequences were aligned to the pdif sequences in the pdifDB database by BLASTN to obtain pdif sequence annotation results in bacterial DNA sequences. The method has great significance for rapidly finding the pdif sequence in the bacterial DNA sequence and determining the drug-resistant gene transfer mechanism mediated by the pdif in the bacteria.
Description
Technical Field
The invention relates to the field of bacterial DNA sequence annotation, in particular to a method and a device for quickly annotating a bacterial pdif sequence, which can be used for analyzing the existence of the pdif in the bacterial DNA sequence and the position of the pdif in the bacterial DNA sequence.
Background
In recent years, the development of Whole Genome Sequencing (WGS) has been rapid. Furthermore, the reduced cost of sequencing increases its likelihood of performing rapid bacterial WGS in the laboratory. The biggest advantage of WGS is that after sequencing results are obtained and assembled, drug resistance genes, mobile elements, etc. of bacteria can be quickly annotated and predicted by a website and command line based tool, greatly accelerating the research progress in the field of molecular characterization of bacteria and epidemiological monitoring.
Bacterial resistance is a major public health problem facing the world. The wide prevalence of drug-resistant bacteria presents a significant challenge to the treatment of infectious diseases. The transmission of intraspecies/interspecies resistance between bacteria can be achieved by moving elements. Understanding the transfer of mobile elements associated with bacterial resistance is crucial to defining the spread of resistance between bacteria. At present, Insertion Sequences (IS), integrants (In), transposons (Transposon, Tn), and the like are mainly reported as mobile elements. In recent years, the XerCD-dif site-specific recombination system is considered as another way for mediating drug-resistant gene transfer, and is more found in Acinetobacter baumannii, which is gradually a hot point of research, but the specific mechanism is still unknown. Many bacteria encode two homologous recombinase proteins, usually occurring in pairs, called XerC/XerD, respectively. XerC/XerD belongs to the family of tyrosine recombinases and can catalyze the cleavage of two consecutive DNA strands and exchange at a defined site dif located in the end region of the chromosome. Generally, the dif site is 28bp in length, and is composed of two Xer binding regions with 11bp inverted repeats at two ends, and the central region is a 6bp spacer region. One monomer of XerC and XerD each bind to a half-binding site of 11bp, XerC binds at the left site and XerD binds at the right site. However, the dif site present on a plasmid is called pdif, and multiple pdif sites, even up to 16, may be present on a single plasmid. Several important drug resistance genes have been found to exist between the pdif sites, such as: blaOXA-24，blaOXA-58，blaOXA-72Tet39, etc. The pdif sites can mediate the transfer of drug resistance genes in the same plasmid and among different plasmids, and especially play a crucial role in the transmission of the drug resistance of acinetobacter baumannii. However, there is no annotation method related to important identification of pdif sites. Therefore, the identification of the pdif locus and the further research on the mechanism of the co-transfer of the gene carrying the drug resistance are of great significance for controlling the transmission of clinically drug-resistant bacteria.
Disclosure of Invention
The invention aims to provide a method and a device for quickly annotating a bacterial pdif sequence, so as to solve the problem that no annotation method related to a pdif locus sequence exists at present.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for rapidly annotating a bacterial pdif, comprising:
constructing a pdifDB database;
obtaining a bacterial DNA sequence to be analyzed through whole genome sequencing;
the bacterial DNA sequences were aligned to the pdif sequences in the pdifDB database by BLASTN to obtain pdif sequence annotation results in bacterial DNA sequences.
Further, constructing a pdifDB database, comprising:
a pdifDB database was extracted and established from the plasmid Genbank file at NCBI.
Further, still include:
and outputting the pdif sequence annotation result in a tabular and graphical form.
Further, the content shown in the graph includes the number of pdifs and the specific positions of pdifs in the bacterial DNA sequence.
Further, the format of the bacterial DNA sequence is Fasta or Genbank format.
In a second aspect, the present invention provides a bacterial pdif rapid annotation device, comprising:
the database construction module is used for constructing a pdifDB database;
the acquisition module is used for acquiring a bacterial DNA sequence to be analyzed through whole genome sequencing;
an alignment module for aligning said bacterial DNA sequence with a pdif sequence in said pdifDB database by BLASTN to obtain pdif sequence annotation results in bacterial DNA sequences.
Through the technical scheme, the invention has the following beneficial effects: firstly, the invention provides a method and a device for quickly annotating bacterial pdif sequences, and the number and the positions of the pdif sequences in bacterial DNA can be annotated only by simply submitting the sequences. Meanwhile, the user can download the annotated related files by himself according to the requirement, and the problem that no related annotation website identified by the pdif sequence exists at present is solved. The user only needs to upload the DNA sequence of the bacteria, and the information about the number and the position of the pdif sequence of the bacteria can be obtained in a very short time after the delivery, and can be further visualized in a graph form. The establishment of the methodology has important significance for researching the transfer mechanism of the drug-resistant gene caused by the pdif sequence in the bacteria.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
FIG. 1 is a flow chart of a method for rapidly annotating a bacterial pdif sequence according to an embodiment of the present invention;
FIG. 2 is a pdif sequence annotation main interface of Acinetobacter baumannii in the examples of the present invention;
FIG. 3a is a schematic diagram showing the result of pdif site analysis of the genomic sequence of Acinetobacter baumannii 255_ n in the example of the present invention;
FIG. 3b is a schematic diagram showing the sequence information of the aligned fragment of Acinetobacter baumannii 255_ n genome in the embodiment of the present invention;
FIG. 4 is a block diagram of a device for fast annotation of bacterial pdif sequences provided in the examples of the present invention.
Detailed Description
For purposes of making the claimed subject matter and the claims more detailed and clear, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings. In addition, the embodiments are only a part of the present application, and not all of the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
FIG. 1 is a flow chart of a method for rapidly annotating bacterial pdif according to an embodiment of the present invention; the method for rapidly annotating bacterial pdif mainly comprises the following steps:
step S101, constructing a pdifDB database;
specifically, the pdifDB database was extracted and established from the annotations in the plasmid Genbank file at the National Center for Biotechnology Information, NCBI. Further, 41 pdif sequences were extracted from the annotations in the plasmid Genbank file on NCBI into the pdifDB database.
Step S102, obtaining a bacterial DNA sequence to be analyzed through whole genome sequencing or directly downloading a complete spliced bacterial DNA sequence from NCBI;
specifically, the sequence to be analyzed and completed with splicing or the already spliced sequence downloaded from the NCBI database is obtained by whole genome sequencing, and the format of the sequence may be Fasta or Genbank, which are the most common formats among DNA sequences.
Step S103, comparing the bacterial DNA sequence with the pdif sequence in the pdifDB database by using a Search Tool (Basic Local Alignment Search Tool, BLAST) based on a Local Alignment algorithm to obtain a pdif sequence annotation result in the bacterial DNA sequence;
specifically, the bacterial DNA sequence was aligned with the pdifDB database constructed in step a by BLAST, and the corresponding pdif in the bacterial DNA sequence was searched, so as to obtain the annotation result of the pdif sequence in the bacterial DNA sequence. Further, before comparison, the minimum coverage and the consistency need to be set, and the common default values are 60% and 90%, respectively. And further uploading the sequence, and comparing the sequence with the pdifDB database established in the step A to obtain the specific position information, the amount and the sequence of the pdif sequence in the bacterial DNA sequence.
Step S104, outputting the pdif sequence annotation result in a form and a graphical form;
specifically, this example will obtain the sequence information of the best matching fragment in the pdifDB database based on the coverage (60%) and consistency (90%) of the alignment, and the system will output the annotation result in a very short time.
Performing visualization operation on the output annotation result, and specifically realizing the visualization operation through the following steps: specifically, a Biopython tool is used for converting the Genbank format into the Genbank format, meanwhile, annotation information is added, then a Python django framework and a nginx server are used for building a website, the result is displayed on a webpage, and GBrows and echarts plug-ins are used for displaying the Genbank format file in a graphical form.
The visualized content mainly comprises the number of pdifs, the sequence of the pdifs and the positions of the pdifs.
In addition, the method can be written into a website through a Python program, the name of the website is pdif, and the flow is as follows:
first, the pdif has one required parameter and four optional parameters. An input file is required, i.e. the assembled bacterial DNA sequence (Fasta or Genbank format). The bacterial DNA sequence loading parameter for Fasta format is-nucleotide (-n), and the bacterial DNA sequence loading parameter for Genbank format is-Genbank (-g). In addition, the sequence file should also include the following optional parameters:
outputting a file directory: - - -resultdir; and (3) referencing a database: -databases; minimum coverage: - -coverage; minimum consistency: -identity.
Next, the corresponding pdif sequence in the bacterial DNA sequence was found by BLAST and comparison with the pdif sequence in the pdifDB database (e value: 1 e-5). The comparison result comprises:
qseqid: inquiring a sequence id number;
sseqid: a target sequence id number;
cadent: percent of matching sequences;
length: matching sequence length;
mismatch: the number of mismatched bases;
gapopen: the number of gaps;
qstart: the starting position of the matched query sequence;
qend: a matched query sequence termination location;
sstart: the starting position of the matched target sequence;
send: (ii) a matched target sequence termination location;
evalue: an expected value;
bitscore: scoring the matching condition;
qseq: nucleic acid sequences of the matched query sequence;
sseq: a nucleic acid sequence of the matched target sequence;
slen: the length of the nucleic acid sequence of the matched query sequence.
Thirdly, based on the comparison coverage rate and consistency, obtaining the sequence information of the best matching fragment; minimum coverage and consistency, default values are 60% and 90%, respectively.
Finally, the result is output in tabular and graphical form for the pdif sequence.
The specific application method of the website is two, one is based on the website, and the other is based on the command line form:
based on the website:
the pdif website was opened and the Fasta or Genbank format file of the target bacterial DNA sequence (fig. 2) was submitted with minimum coverage and consistency, defaults of 60% and 90%, respectively. The sequence of deliveries, the program was run for <1 min/time.
The analysis results will mainly show the specific information of the pdif of the bacterial DNA sequence. As shown in FIG. 3, the test strain was Acinetobacter baumannii 255_ n, which revealed that the strain had 8 pdifs (FIG. 3a), and the positions of the pdif sequences in the bacterial DNA sequence were clearly seen (FIG. 3 b).
Based on the command line:
the method has been uploaded to GitHub as follows:
this script accepts sequence files in Fasta or Genbank format (plasmid sequence less than 1M);
the output file includes: amrgene.txt, isfider.filter.xls, pdif _ site.txt, plasma.html;
the process comprises the following steps:
firstly, searching for a drug-resistant gene of a bacterial DNA sequence, and if not, terminating the program;
secondly, based on the pdif database, all pdifs are searched, if not, the procedure is terminated;
further, looking for paired pdifs, only keeping paired pdifs, if not, the procedure is terminated;
and finally, outputting the result in a chart form. In the figure, blue represents the drug resistance gene, gray line represents pdif, and green represents the insertion element.
Operation:
the pdiffender is a python3.X script, is easy to use and supports Window and Linux systems;
first, BLAST is required, which should be added to the environmental variables;
second, read Requirements. txt, check for dependent items or run directly: pip install-r requisitions.txt;
finally, the command is run as follows:
python pdiffield, py-i fastfile-o outdi or python pdiffield, py-g genbank file-o outdi
Example 2:
the embodiment of the present invention provides a bacterial pdif fast annotation device (as shown in fig. 4), which can perform a bacterial pdif fast annotation method provided by any embodiment of the present invention. The method has the corresponding functional modules and beneficial effects. The device includes:
a database construction module 21, configured to construct a pdifDB database;
an obtaining module 22, configured to obtain a bacterial DNA sequence to be analyzed by whole genome sequencing;
an alignment module 23 for aligning the bacterial DNA sequence with the pdif sequences in the pdifDB database by BLAST to obtain pdif sequence annotation results in bacterial DNA sequences.
Further comprising: and the output module 24 is used for outputting the pdif sequence annotation result in a tabular and graphical form.
In the embodiments of the present invention, the corresponding descriptions are focused, and the related descriptions of other embodiments may be referred to for parts that are not described in detail in a certain embodiment.
In the embodiments provided in the present application, the disclosed technical content can be implemented in other ways. The above-mentioned embodiments are merely illustrative, and for example, the division of the units may be a logical division, and in actual implementation, there may be another division, for example, multiple units or components may be combined/integrated into another system, or some features may be omitted, or not executed. The units described as separate parts are not physically separate and may be located in one unit or distributed in a plurality of units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of the embodiment. In addition, the integrated unit may be implemented in a hardware form/a software form.
The integrated unit, if implemented in software and sold or used as a stand-alone product, may be stored in a computer-readable storage medium. Wherein the storage medium includes: a removable hard disk, a usb disk, a Random Access Memory (RAM), a Read-Only Memory (ROM), or an optical disk.
The above description is only exemplary of the invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the invention may be included within the scope of the invention.
Claims (10)
1. A bacterial pdif rapid annotation method is characterized by comprising the following steps:
constructing a pdifDB database;
obtaining a bacterial DNA sequence to be analyzed through whole genome sequencing;
the bacterial DNA sequences were aligned to the pdif sequences in the pdifDB database by BLASTN to obtain pdif sequence annotation results in bacterial DNA sequences.
2. The method for rapid annotation of bacterial pdif according to claim 1, wherein the pdifDB database is constructed by:
a pdifDB database was extracted and established from the plasmid Genbank file at NCBI.
3. The method for rapid annotation of bacterial pdif according to claim 1, further comprising:
and outputting the pdif sequence annotation result in a tabular and graphical form.
4. The method for fast annotation of bacterial pdif according to claim 3, wherein the graphical representation comprises the number of pdif and the specific location of pdif in the bacterial DNA sequence.
5. The method for rapidly annotating bacterial pdif according to claim 1, wherein said bacterial DNA sequence is in Fasta or Genbank format.
6. A bacterial pdif rapid annotation device, comprising:
the database construction module is used for constructing a pdifDB database;
the acquisition module is used for acquiring a bacterial DNA sequence to be analyzed through whole genome sequencing;
an alignment module for aligning said bacterial DNA sequence with a pdif sequence in said pdifDB database by BLASTN to obtain pdif sequence annotation results in bacterial DNA sequences.
7. The bacterial pdif rapid annotation device of claim 6, wherein the pdifDB database is constructed by:
a pdifDB database was extracted and established from the plasmid Genbank file at NCBI.
8. The bacterial pdif rapid annotation device of claim 6, further comprising:
and the output module is used for outputting the pdif sequence annotation result in a tabular and graphical form.
9. The apparatus for rapid annotation of bacterial pdif according to claim 8, wherein the graphical representation comprises the number of pdif and the specific location of pdif in the bacterial DNA sequence.
10. The device for rapidly annotating bacterial pdif according to claim 6, wherein said bacterial DNA sequence is in Fasta or Genbank format.
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Application publication date: 20210202 |