CN112687343A - Nanopore sequencing-based broad-spectrum pathogenic microorganism and drug resistance analysis system - Google Patents
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Abstract
The invention provides a nanopore sequencing-based broad-spectrum pathogenic microorganism and drug resistance analysis system, which integrates various analysis programs, has a simple and clear operation interface, can be easily operated by an experimenter in a short time, can generate a graphical analysis report by one key, solves the problems of complex visual display and difficult operation of the conventional analysis process, realizes the visual display of the analysis process, optimizes parameter adjustment input and realizes the effect of analyzing a sequencing result by one key.
Description
Technical Field
The invention relates to a gene analysis system, in particular to a nanopore sequencing-based broad-spectrum pathogenic microorganism and drug resistance analysis system.
Background
In recent years, the threat to public health has been increasing due to the prevalence of infectious diseases caused by viral microorganisms. According to the world health statistics report, over 1000 million people die from infectious diseases every year worldwide, accounting for about 30% of the total death rate. The major causes of mortality in viral infections are high: firstly, the infectivity is strong, and secondly, the microbial species of infectious diseases are increasingly complex. The threat of common pathogenic microorganisms is not eliminated, but drug-resistant strains appear, and the appearance of new pathogens brings great difficulty to clinical diagnosis and treatment.
At present, the early and rapid detection of pathogenic microorganisms is the key for infectious disease prevention and control, and the conventional method is to detect drug resistance genes and virulence genes of the pathogenic microorganisms through metagenomic sequencing of second-generation sequencing, but a large amount of original data generated by the currently adopted sequencing method needs to be analyzed by professional experimenters trained for a long time, and the professional experimenters need to call various programs in a linux shell command line form to realize analysis work such as filtration, sequence comparison, microorganism species classification, microorganism reading number statistics, pathogenic microorganism detection, target species data extraction, genome integrity calculation and the like on the original sequences. Such a drawback lies in that it is necessary for professional experimenters to have very strong biological information analysis and linux system operation capabilities, and each analysis program has different selection schemes and parameters, and professional experimenters are required to spend a large amount of time to repeatedly search and adjust the programs and parameters, so that the efficiency is very low, the visualization effect display of data is problematic, and the degree of automation is very low.
In summary, no simple and easy-to-operate sequencing analysis system for detecting resistance toxicity of broad-spectrum microorganisms exists, so that the progress of pathogenic microorganism sequencing work in public health laboratories is limited due to the lack of professional bioanalyzers and high-quality pathogenic databases in public health experimental processes such as hospitals, disease control and the like.
Disclosure of Invention
The invention aims to provide a nanopore sequencing-based broad-spectrum pathogenic microorganism and drug resistance analysis system, which integrates various analysis programs, has a simple and clear operation interface, can be easily operated by experimenters in a short time, can generate a graphical analysis report by one key, and solves the problems of complex visual display and difficult operation of the conventional analysis process.
In order to achieve the above objects, the present technical solution provides a nanopore sequencing-based analysis system for broad-spectrum pathogenic microorganisms and drug resistance, comprising: the system comprises a data analysis system and a sample management system which are mutually associated, wherein the data analysis system is used for acquiring sequencing data of pathogenic microorganisms to be detected and analyzing the sequencing data, the sample management system is used for acquiring sample information corresponding to the pathogenic microorganisms to be detected, and the sequencing data and the sample information are associated; the data analysis system includes: the task establishing unit is used for establishing an analysis task corresponding to sequencing data of the pathogenic microorganism to be detected, wherein the analysis task stores the sequencing data and analysis parameters of the pathogenic microorganism to be detected; a pathogenic microorganism database which comprises a species database for storing a plurality of broad-spectrum pathogenic microorganisms and a functional gene database for storing functional genes; the sequence comparison unit is used for obtaining a comparison instruction, comparing the sequencing data with the species database to obtain a species sequence of the pathogenic microorganism to be detected, and comparing the sequencing data with the functional gene database to obtain a functional gene sequence; the species sequence analysis unit is used for acquiring a species analysis instruction and performing at least one sequence analysis task of microorganism species classification, microorganism reading number statistics, target sequence extraction and genome integrity calculation based on a species sequence; the functional gene sequence analysis unit is used for acquiring a gene analysis instruction and carrying out function identification based on the functional gene sequence; and the analysis report generating unit is used for acquiring the report command, extracting the analysis result data of the species sequence analyzing unit or the functional gene sequence analyzing unit and generating an analysis report.
Compared with the prior art, the technical scheme has the following characteristics and beneficial effects: providing visual display of an analysis process, optimizing parameter adjustment input, and analyzing a sequencing result in a one-click mode; integrating analysis processes, solving the problem of data analysis which cannot be processed by the traditional analysis system through self-programming, and realizing automatic sequencing data analysis; the analysis system which provides a graphical interface in one key mode and the PDF format detection report in one key mode enable data interpretation of a sequencing sequence to be simpler.
Drawings
FIG. 1 is a schematic block diagram of a broad-spectrum pathogenic microorganism and drug resistance analysis system based on nanopore sequencing according to the present invention.
FIG. 2 is a schematic diagram of input data and analysis parameters.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
It is understood that the terms "a" and "an" should be interpreted as meaning that a number of one element or element is one in one embodiment, while a number of other elements is one in another embodiment, and the terms "a" and "an" should not be interpreted as limiting the number.
The integrated analysis system establishes a complete analysis process aiming at sequencing data of the broad-spectrum pathogenic microorganisms, and performs overall analysis of quality control, sequence comparison, microorganism species classification, microorganism reading number statistics, pathogenic microorganism detection, target species data extraction and genome integrity calculation aiming at sequencing data of second-generation sequencing and third-generation sequencing. In addition, the whole analysis process of the sequencing analysis is visually displayed, an operator can simply perform analysis operation according to an operation instruction on an operation interface, and an analysis result is comprehensively displayed in a chart form.
The scheme content of the nanopore sequencing-based broad-spectrum pathogenic microorganism and drug resistance analysis system comprises the following steps: the sequencing data of the drug-resistant virulence genes of unknown pathogenic microorganisms based on nanopore sequencing construct a comprehensive analysis process, a high-quality pathogenic microorganism database with complete pathogenic microorganism species and complete pathogen annotation is built, the pathogenic microorganisms can be rapidly identified, key functional genes such as the drug-resistant genes and virulence genes can be identified, and genome assembly and traceability analysis can be performed if the pathogenic microorganisms have sufficient data; in addition, the analysis system supports a nanopore sequencing technology with complete data types and data formats of fastq and fast5, can analyze multi-sample data simultaneously, and can also support single-end and double-end fastq second-generation data; the analysis process is visualized, and the analysis result can be generated one key and displayed in a graphical mode.
Specifically, a wide-spectrum pathogenic microorganism and drug resistance analysis system based on nanopore sequencing is provided with a plurality of sequencing channels, can complete 200Mb fastq-format nanopore sequencing data biological information analysis within 10 minutes, and can be matched with all high-throughput sequencing platforms such as Illumina, Huada, Ion Torrent and Pacbio to support simultaneous analysis of nanopore sequencing data of 96 samples at most.
Fig. 1 shows a schematic diagram of a framework of a nanopore sequencing-based broad-spectrum pathogenic microorganism and drug resistance analysis system of the present embodiment, which can rapidly identify a pathogenic microorganism to be detected and also identify key functional genes of the pathogenic microorganism, and the system includes:
the system comprises a data analysis system and a sample management system which are mutually associated, wherein the data analysis system is used for acquiring sequencing data of pathogenic microorganisms to be detected and analyzing the sequencing data, the sample management system is used for acquiring sample information corresponding to the pathogenic microorganisms to be detected, and the sequencing data and the sample information are associated;
the data analysis system includes:
the task establishing unit is used for establishing an analysis task corresponding to sequencing data of the pathogenic microorganism to be detected, wherein the analysis task stores the sequencing data and analysis parameters of the pathogenic microorganism to be detected;
a pathogenic microorganism database which comprises a species database for storing a plurality of broad-spectrum pathogenic microorganisms and a functional gene database for storing functional genes;
the sequence comparison unit is used for obtaining a comparison instruction, comparing the sequencing data with the species database to obtain a species sequence of the pathogenic microorganism to be detected, and comparing the sequencing data with the functional gene database to obtain a functional gene sequence;
the species sequence analysis unit is used for acquiring a species analysis instruction and performing at least one sequence analysis task of microorganism species classification, microorganism reading number statistics, target sequence extraction and genome integrity calculation based on a species sequence;
the functional gene sequence analysis unit is used for acquiring a gene analysis instruction and carrying out function identification based on the functional gene sequence;
and the analysis report generating unit is used for acquiring the report instruction, abstracting the analysis result data of the species sequence analyzing unit or the functional gene sequence analyzing unit and generating a visual analysis report.
In the scheme, the nanopore sequencing-based broad-spectrum pathogenic microorganism drug-resistant toxicity integrated analysis system can be used for identifying drug-resistant toxicity genes of unknown pathogenic microorganisms. Therefore, the functional genes of the scheme comprise drug resistance genes and virulence genes, and correspondingly, various drug resistance genes and various virulence genes are stored in the functional gene database. And after the sequence comparison unit acquires the comparison instruction, comparing whether the sequencing data of the pathogenic microorganism to be detected has a corresponding functional gene sequence, and if so, judging that the pathogenic microorganism to be detected has a functional gene. It is worth mentioning that the functional genes stored in the functional gene database can be replaced according to actual requirements.
The task establishing unit is provided with a plurality of interfaces aiming at different types of data, different sequencing analysis channels are arranged corresponding to the different interfaces, and the corresponding sequencing analysis channels are selected according to the types of the sequencing data. Specifically, the multiple interfaces of the task establishing unit in the scheme enable the analysis system to analyze not only fastq and fast5 types of nanopore sequencing data, but also single-end and double-end fastq second-generation nanopore sequencing data, and simultaneously analyze and process multiple types of sequencing data, and the analysis system is applicable to various types of sequencing data, such as: illumina, huada, ion torrent, Pacbio, etc. are almost all high throughput sequencing platforms. This is because the present solution classifies the tasks created by the task creating unit and individually sets the sequencing analysis channel.
Specifically, the task establishing unit comprises a second-generation sequencing task module and a third-generation sequencing task module, the second-generation sequencing task module stores a second-generation sequencing task and corresponding analysis parameters, the third-generation sequencing task module stores a third-generation sequencing task and corresponding analysis parameters, the task establishing unit is provided with a parameter setting module, the parameter setting module is used for manually adjusting the parameters of sequencing data, and the parameter setting module is displayed on a system interface through a visual process. Moreover, it is worth mentioning that an independent sequencing analysis channel is established for each sequencing task, and a storage folder is established for the corresponding sequencing task.
Specifically, the analysis parameters for the third-generation sequencing task include a task name, a mode, a sequence path, a sample mixing reagent, a thread number, a length limit value and an accuracy limit value, wherein the task name defines the name of each sequencing analysis channel so as to facilitate the user to quickly position and manage the established sequencing data; the mode can select one of fast5, fast q and barcodefastwq, and different subsequent analysis channels are selected for each mode; inputting a file path of the folder by the sequence path; the mixed sample reagent provides single sample sequencing and a sequencing scheme of a multi-sample sequencing reagent corresponding to the Nanopore sequencing according to the analysis type.
The analysis parameters aiming at the second-generation sequencing task comprise a task name, sequence selection and thread number, and the mode corresponding to the second-generation sequencing task is a fastq mode.
Particularly, the analysis parameters of the sequencing task can be set by manual selection according to the scheme. In particular, the pattern of sequencing data corresponds to different subsequent sequencing analysis efforts. The default of the thread number is 10, the default of the length limit value is 500, the default of the accuracy limit value is 80, and the specific parameters can be correspondingly adjusted according to the parameter setting module. In particular, since the present protocol provides a separate sequencing analysis channel, it allows the protocol to be targeted to different types of data.
The user inputs sequencing data on an interface of the analysis system and fills or modifies corresponding analysis parameters according to the instructions, the task establishing unit establishes corresponding storage folders based on the obtained sequencing data and the analysis parameters, and if the input sequencing data is a third-generation sequencing task, options of different data models are displayed.
Sample information corresponding to sequencing data is input into the sample management system, and the sample information includes but is not limited to: the task name of the sequencing data, the sampling information of the sequencing data and the personnel information of the sampling personnel corresponding to the sequencing data. The sampling information includes sample type, sampling date, and sequencing date. The person information includes the name, gender, and age of the person who sampled. And associating the sample information with the sequencing data, and storing the sample information and the sequencing data in a folder corresponding to the sequencing data.
And filling sample information on an interface of the analysis system by a user according to the instruction, and associating the task name in the task establishing unit with the option corresponding to the task name of the sequencing data for the user to select autonomously. Alternatively, the user may enter "the task name of the sequencing data" to match the corresponding sequencing data from the task creation unit.
And when the corresponding sequencing data are stored in the folder for storing the sequencing data by the analysis system, carrying out subsequent sequencing analysis according to the operation instruction of the user. The scheme reconfigures the triggering interfaces and the cascade relations of a pathogenic microorganism database, a sequence comparison unit, a species sequence analysis unit, a functional gene sequence analysis unit and an analysis report generation unit according to a sequence analysis process, wherein the cascade relations are as follows: the species sequence analysis unit and the functional sequence analysis unit are subordinate associated task nodes of the sequence comparison unit, the pathogenic microorganism database and the task establishment unit are peer associated task nodes of the sequence comparison unit, and the analysis report generation unit is subordinate associated task nodes of the sequence comparison unit, the species sequence analysis unit and the functional gene sequence analysis unit. A trigger interface of the sequence comparison unit corresponds to the comparison instruction, and the sequence comparison unit is triggered only after the comparison instruction is obtained; the trigger interfaces of the species sequence analysis unit and the functional gene sequence analysis unit correspond to the analysis instruction and the comparison result of the sequence comparison unit, and the species sequence analysis unit and the functional gene sequence analysis unit are triggered only after the analysis instruction and the corresponding comparison result are obtained.
The operation pressure of the analysis system is reduced through the arrangement of the mode, and the operation difficulty of operators is reduced. An operator selects corresponding content on an application interface of the analysis system according to requirements, generates corresponding instructions, cannot trigger lower task nodes under the condition that higher cascade conditions are not met due to the fact that cascade relations are set among units of the analysis system, and data to be analyzed are circulated in the analysis system according to the set flow direction.
The comparison instruction of the sequence comparison unit can be a button displayed on a display interface, the comparison instruction comprises a species sequence comparison instruction and a functional gene comparison instruction, if the species sequence comparison instruction is obtained, the species database is triggered to carry out comparison, the comparison result is a species comparison result, and the species sequence analysis unit carries out species sequence analysis only after the species comparison result and the analysis instruction are obtained; if the functional group comparison instruction is obtained, the functional gene database is triggered to carry out comparison, the comparison result is a functional gene comparison result, and the functional gene analysis unit carries out functional gene analysis only after the functional gene comparison result and the analysis instruction are obtained. And the comparison mode adopts a matching mode, if the sequence is obtained by matching in the species database or the functional gene database, the sequencing data can be determined to contain a corresponding sequence, and the corresponding species sequence or the functional gene is selected.
Because the processing procedures of the sequence alignment unit in the scheme are independent and related, the result generated by alignment can be directly extracted in the analysis report generation unit. In addition, since the task establishing unit classifies the data modes, the sequence comparison unit can normally operate according to the comparison of the corresponding modes.
In some embodiments, the sequence alignment unit performs a screening filter on the sequencing data followed by an alignment of the sequences, the screening filter screening noise segments in the sequencing data.
The species sequence analysis unit can be divided into an independent microorganism species classification unit, a microorganism reading number statistical unit, a target sequence extraction unit and a genome integrity calculation unit according to the analysis content, and one or more species classification units, microorganism reading number statistical units, target sequence extraction units and genome integrity calculation units are triggered according to the analysis instruction, so that the simple operation analysis of the whole process is realized. That is to say, the present solution gathers the tasks of species sequence analysis, and the operator selects the tasks of analysis according to the requirement, and triggers the corresponding species sequence analysis unit to perform analysis. The microorganism species classification unit classifies the pathogenic microorganisms to be detected based on species sequences of the pathogenic microorganisms to be detected; a target sequence extraction unit for extracting a target sequence from a species sequence of a pathogenic microorganism to be detected; the microorganism reading number counting unit counts the reading number of the species sequence of the pathogenic microorganism to be detected; and the genome integrity calculation unit is used for calculating the integrity of the species sequence gene of the pathogenic microorganism to be detected.
Correspondingly, the user selects an analysis task on a page corresponding to the analysis system according to the classification of microorganism species, the statistics of microorganism reading number, the extraction of target sequence and the calculation of genome integrity, and correspondingly generates different species sequence analysis results. Specifically, the analysis tasks of each analysis unit are conventional, and the scheme is characterized in that a plurality of analysis tasks are integrated, and the integration is based on that a sequencing analysis channel is independently arranged according to the mode of sequencing data and the cascade relation among the units is set.
In addition, the scheme additionally comprises a pathogen annotation unit for storing pathogen annotation information aiming at the species sequence, and the pathogen annotation information is stored in a file library for storing sequencing data. In some embodiments, the pathogenic annotation information is entered manually; in other embodiments, the analysis unit is embedded with a pathogen database, and the pathogen annotation information is obtained by comparing the obtained species classifications.
At this time, the pathogen database is a lower-level association unit of the microorganism species classification unit, and the trigger interface of the pathogen database generates pathogen annotation information corresponding to an analysis task of microorganism species classification and a pathogen annotation instruction.
Functional identification of functional gene sequence analysis units includes classification of drug resistance elements and confirmation of drug resistance mechanisms.
In addition, the analysis report generating unit embeds an analysis report template, extracts corresponding data content according to the gene analysis command and fills the data content into the analysis report template, wherein the extracted data content comprises: one or more of sample information, species sequence analysis result, drug resistance element type and pathogen annotation information. Moreover, it should be noted that, since the processing procedures of the present solution are independent and related, the analysis report generation unit is convenient to extract the corresponding content independently. In addition, the analysis report generation unit can generate a chart-type analysis report according to the extracted data content.
The flow interface of the analysis system provided by the scheme is simple and easy to operate, after the sequencing data and the sample information are input by an operator, the corresponding analysis content is displayed according to the indication, and finally the analysis content is summarized to obtain an analysis report, so that one-click output from offline data biological information to a result report is realized.
The system for integrally analyzing drug resistance and toxicity of broad-spectrum pathogenic microorganisms based on nanopore sequencing provided by the scheme can be carried on a computer system to run, and the computer system of the server comprises a Central Processing Unit (CPU) which can execute various appropriate actions and processes according to programs stored in a Read Only Memory (ROM) or programs loaded from a storage part into a Random Access Memory (RAM). In the RAM, various programs and data necessary for system operation are also stored. The CPU, ROM, and RAM are connected to each other via a bus. An input/output (I/O) interface is also connected to the bus. The modules described in the embodiments of the present invention may be implemented by software, or may be implemented by hardware, and the described modules may also be disposed in a processor.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.
Claims (10)
1. A nanopore sequencing-based broad-spectrum pathogenic microorganism and drug resistance analysis system is characterized by comprising:
the system comprises a data analysis system and a sample management system which are mutually associated, wherein the data analysis system is used for acquiring sequencing data of pathogenic microorganisms to be detected and analyzing the sequencing data, the sample management system is used for acquiring sample information corresponding to the pathogenic microorganisms to be detected, and the sequencing data and the sample information are associated;
the data analysis system includes: the task establishing unit is used for establishing an analysis task corresponding to sequencing data of the pathogenic microorganism to be detected, wherein the analysis task stores the sequencing data and analysis parameters of the pathogenic microorganism to be detected; a pathogenic microorganism database which comprises a species database for storing a plurality of broad-spectrum pathogenic microorganisms and a functional gene database for storing functional genes; the sequence comparison unit is used for obtaining a comparison instruction, comparing the sequencing data with the species database to obtain a species sequence of the pathogenic microorganism to be detected, and comparing the sequencing data with the functional gene database to obtain a functional gene sequence; the species sequence analysis unit is used for acquiring a species analysis instruction and performing at least one sequence analysis task of microorganism species classification, microorganism reading number statistics, target sequence extraction and genome integrity calculation based on a species sequence; the functional gene sequence analysis unit is used for acquiring a gene analysis instruction and carrying out function identification based on the functional gene sequence; and the analysis report generating unit is used for acquiring the report command, extracting the analysis result data of the species sequence analyzing unit or the functional gene sequence analyzing unit and generating an analysis report.
2. The nanopore sequencing-based analysis system for broad-spectrum pathogenic microorganisms and drug resistance according to claim 1, wherein the task establishment unit comprises a second-generation sequencing task module and a third-generation sequencing task module, the second-generation sequencing task module stores a second-generation sequencing task and corresponding analysis parameters, the third-generation sequencing task module stores a third-generation sequencing task and corresponding analysis parameters, and the task establishment unit is provided with a parameter setting module.
3. The nanopore sequencing-based pathogenic microorganism drug-resistant virulence gene integrated analysis system of claim 2, wherein an independent sequencing analysis channel is established for each sequencing task, and a storage folder is established for the corresponding sequencing task, with sample information associated with the folder.
4. The nanopore sequencing-based analysis system for broad spectrum pathogenic microorganisms and drug resistance according to claim 2, wherein the analysis parameters for the third generation sequencing task include task name, mode, sequence path, sample mixing reagent, thread number, length limit value and accuracy limit value, and the mode can be selected from one of fast5, fast q and barcoded fast q; the analysis parameters aiming at the second-generation sequencing task comprise a task name, sequence selection and thread number, and the mode corresponding to the second-generation sequencing task is a fastq mode.
5. The nanopore sequencing-based pathogenic microorganism drug-resistant virulence gene integrated analysis system of claim 1, wherein a pathogenic microorganism database, a sequence alignment unit, a species sequence analysis unit, a functional gene sequence analysis unit, a trigger interface of an analysis report generation unit, and a cascade relationship are configured according to a sequence analysis process.
6. The nanopore sequencing-based analysis system for broad spectrum pathogenic microorganisms and drug resistance as claimed in claim 5, wherein the trigger interfaces of the species sequence analysis unit and the functional gene sequence analysis unit correspond to the analysis command and the comparison result of the sequence comparison unit, and trigger the species sequence analysis unit and the functional gene sequence analysis unit when the analysis command and the corresponding comparison result are obtained.
7. The nanopore sequencing-based broad spectrum pathogenic microorganism and drug resistance analysis system of claim 1, comprising a pathogen annotation unit storing pathogen annotation information for a species sequence.
8. The nanopore sequencing-based broad spectrum pathogenic microorganism and drug resistance analysis system of claim 7, wherein a pathogen database is built in, and the trigger interface of the pathogen database corresponds to the analysis task of microorganism species classification and the pathogen annotation command.
9. The nanopore sequencing-based broad spectrum pathogenic microorganism and drug resistance analysis system of claim 1, wherein the analysis report generation unit is internally provided with an analysis report template, extracts corresponding data content according to the gene analysis instruction and fills the data content into the analysis report template, and the extracted data content comprises: one or more of sample information, species sequence analysis result, drug resistance element type and pathogen annotation information.
10. The nanopore sequencing-based broad spectrum pathogenic microorganism and drug resistance analysis system of claim 9, wherein the functional genes are drug resistance genes and virulence genes.
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