16srna blue-green algae high-throughput sequencing method and system

Abstract

The invention discloses a 16srna cyanobacteria high-throughput sequencing method and system, which are characterized in that: by using NCBI and SILVA databases, a cyanobacteria 16srna gene sequence is widely compared and excavated through a BLAST tool, and a conservative sequence region with cyanobacteria specificity is identified; based on a mining result, designing a plurality of pairs of candidate amplification primers by adopting Primer3 and Oligo primer design software; evaluating the specificity, sensitivity and product quality of candidate primers through an in-vitro PCR amplification experiment, and screening out an optimal primer pair; selecting a high-throughput sequencing platform according to experimental requirements; evaluating the optimal sequencing depth by using simulation data or known community data; the method has the beneficial effects that by designing the amplification primers with enhanced specificity and optimizing a sequencing strategy, high-coverage sequencing is realized, an efficient and automatic bioinformatics analysis process is developed, and a standardized species annotation and community structure analysis system is constructed, so that the analysis precision and efficiency as well as the comparability and repeatability of results are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of cyanobacteria sequencing, and in particular relates to a 16S RNA cyanobacteria high-throughput sequencing method and system. Background Art

[0002] As an important group of prokaryotes, cyanobacteria are increasingly attracting attention for their diversity and role in the ecosystem; however, traditional cyanobacterial 16S RNA gene sequence analysis methods are limited in sequence coverage, classification accuracy and the ability to discover new species; with the rapid development of high-throughput sequencing technology and the widespread application of deep learning algorithms, it is possible to build a more accurate cyanobacterial sequence database.

[0003] Disadvantages of existing technology:

[0004] Data redundancy and inconsistency: Existing cyanobacterial 16S RNA gene amplicon sequence databases often have problems such as data redundancy, uneven sequence quality, and inconsistent species naming, which lead to bias in data analysis and comparison;

[0005] Resolution and accuracy: Due to the conservation and variability of 16S RNA gene sequences of cyanobacteria, the resolution and accuracy of existing databases in species identification are limited, especially for species with close genetic relationships.

[0006] Update lag: With the popularization of high-throughput sequencing technology and the acceleration of scientific research progress, new cyanobacterial species and sequences continue to emerge, but the update speed of existing databases often lags behind scientific research needs and cannot reflect the latest research results in a timely manner;

[0007] Lack of integrated solutions: Existing database systems often only focus on the storage and retrieval of sequence data and lack integration with bioinformatics analysis tools, which requires users to spend a lot of time and energy on data processing and analysis;

[0008] Unfriendly user interface: The user interface design of some existing database systems is not intuitive and friendly enough, and user operations are complicated, which is not conducive to use and promotion by non-professionals. Summary of the invention

[0009] The purpose of the present invention is to provide a 16S RNA cyanobacteria high-throughput sequencing method and system, which significantly improves the analysis accuracy, efficiency and comparability and repeatability of the results by designing specificity-enhanced amplification primers, optimizing sequencing strategies to achieve high-coverage sequencing, developing efficient and automated bioinformatics analysis processes, and constructing a standardized species annotation and community structure analysis system.

[0010] To achieve the above object, the present invention provides the following technical solution: a 16S RNA high-throughput sequencing method for cyanobacteria, comprising:

[0011] Specific amplification primer design and verification:

[0012] Sequence database mining: Using the NCBI and SILVA databases, the BLAST tool was used to conduct extensive comparison and mining of cyanobacterial 16sRNA gene sequences to identify conservative and cyanobacterial-specific sequence regions;

[0013] Primer design: Based on the mining results, Primer3 and Oligo primer design software were used to design multiple pairs of candidate amplification primers;

[0014] Primer validation: Through in vitro PCR amplification experiments, the specificity, sensitivity and product quality of candidate primers are evaluated to screen out the optimal primer pair;

[0015] Optimize sequencing strategy and library construction:

[0016] Sequencing platform selection: Select a high-throughput sequencing platform based on experimental requirements;

[0017] Sequencing depth and coverage assessment: Use simulated data or known community data to assess the optimal sequencing depth;

[0018] Library construction: The 16S RNA gene of the cyanobacteria sample was amplified using optimized PCR conditions, and a high-quality sequencing library was constructed after purification, quantification, and end repair;

[0019] Efficient and automated bioinformatics analysis:

[0020] Data quality control: FastQC and Trim Galore tools were used to remove low-quality sequences and contamination;

[0021] Sequence de-redundancy and clustering: CD-HIT and DADA2 tools were used to de-redundant and cluster into OTUs;

[0022] Species annotation and classification: compare the OTU representative sequences with the authoritative database for species annotation;

[0023] Community structure analysis: QIIME 2 and Mothur software were used to calculate diversity index and distance matrix and analyze community structure;

[0024] Standardized result output and report generation:

[0025] Standardized result output: Output the analysis results in a standardized format to ensure comparability;

[0026] Report Generation: Generate detailed reports using programming languages ​​and graphical tools.

[0027] As a preferred technical solution of the present invention, experimental requirements include sequencing depth, throughput, and cost.

[0028] As a preferred technical solution of the present invention, the high-throughput sequencing platform includes Illumina and IonTorrent.

[0029] As a preferred technical solution of the present invention, in designing multiple pairs of candidate amplification primers, the primer design takes into account melting temperature, GC content, secondary structure formation potential and complementarity between primers.

[0030] As a preferred technical solution of the present invention, the OTU representative sequence is compared with the authoritative database, and the authoritative database includes SILVA and Greengenes.

[0031] As a preferred technical solution of the present invention, the diversity index includes Shannon index and Simpson index.

[0032] As a preferred technical solution of the present invention, in generating a detailed report using programming languages ​​and graphical tools, R language and Python programming languages ​​are used in combination with graphical tools ggplot2 and seaborn to generate a detailed technical report or scientific paper containing analysis results, charts and discussions.

[0033] As a preferred technical solution of the present invention, the optimized PCR conditions include denaturation temperature, annealing temperature, extension temperature, and cycle number.

[0034] The present invention also discloses a 16S RNA cyanobacteria high-throughput sequencing system, comprising

[0035] Specific amplification primer design module:

[0036] Database interface: connect to NCBI and SILVA databases to mine cyanobacteria 16srna gene sequences;

[0037] Primer design software: integrates Primer3 and Oligo software to automatically design and screen specific amplification primers;

[0038] Experimental verification platform: supports in vitro PCR amplification experiments to verify primer specificity and efficiency;

[0039] Optimize sequencing strategy and library construction modules:

[0040] Sequencing platform selection tool: recommend the optimal sequencing platform based on experimental requirements;

[0041] Sequencing depth assessment model: Evaluate the optimal sequencing depth based on simulation or known data;

[0042] Library construction automated workstation: automated equipment that integrates PCR amplification, purification, quantification, and library construction steps;

[0043] Efficient and automated bioinformatics analysis module:

[0044] Data quality control software: Integrates FastQC and Trim Galore tools to automatically remove low-quality sequences and contamination;

[0045] Sequence processing and analysis platform: supports CD-HIT, DADA2 de-redundancy, BLAST, RDP Classifier species annotation, QIIME 2, Mothur community structure analysis;

[0046] Result visualization tools: Integrate ggplot2 and seaborn graphical tools to generate intuitive analysis charts;

[0047] Standardized result output and report generation module:

[0048] Standardized result output module: automatically generates analysis results in a standardized format;

[0049] Report Generator: A combination of programming language and templates to quickly generate technical reports or scientific papers containing analysis results, charts and discussions.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] By designing specificity-enhanced amplification primers, optimizing sequencing strategies to achieve high-coverage sequencing, developing efficient and automated bioinformatics analysis processes, and building a standardized species annotation and community structure analysis system, the analysis accuracy, efficiency, and comparability and repeatability of the results can be significantly improved;

[0052] High-throughput sequencing technology can process a large number of samples at the same time, greatly improving sequencing efficiency and making it possible to obtain a large number of cyanobacterial 16SRNA gene sequences in a short period of time;

[0053] Ability to detect microorganisms at very low abundance in samples, helping to discover rare or understudied cyanobacterial species;

[0054] With the continuous development and popularization of sequencing technology, the cost of sequencing has gradually decreased, making it possible for more research institutions and individuals to afford the cost of 16S RNA gene sequencing of cyanobacteria;

[0055] 16SRNA gene sequencing technology does not require the isolation and cultivation of microorganisms. DNA can be directly extracted from environmental samples for sequencing, avoiding the deviations that may be introduced during the cultivation process.

[0056] It is not only suitable for cyanobacteria, but also for the diversity analysis of other microorganisms such as bacteria and fungi, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The flowchart of the sequencing method of the present invention is shown in FIG. DETAILED DESCRIPTION

[0058] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] Example 1

[0060] See also Figure 1 , which is the first embodiment of the present invention, and provides a 16S RNA cyanobacteria high-throughput sequencing method, comprising:

[0061] 1. Design and verification of specific amplification primers:

[0062] Sequence database mining: Using authoritative databases such as NCBI and SILVA, and using tools such as BLAST, we conducted extensive comparisons and in-depth mining of cyanobacterial 16SRNA gene sequences to identify highly conservative and cyanobacterial-specific sequence regions;

[0063] Primer design: Based on the mining results, primer design software such as Primer3 and Oligo are used to design multiple pairs of candidate amplification primers. Primer design needs to consider factors such as melting temperature (Tm), GC content, secondary structure formation potential, and complementarity between primers to ensure primer specificity and amplification efficiency.

[0064] Primer verification: The candidate primers were verified for specificity through in vitro PCR amplification experiments; known cyanobacteria samples and non-cyanobacteria control samples were used to evaluate the amplification specificity, sensitivity and quality of the amplified products of the primers, and finally the primer pairs with high specificity and good amplification efficiency were selected for subsequent experiments;

[0065] 2. Optimize sequencing strategy and library construction:

[0066] Sequencing platform selection: Select a suitable high-throughput sequencing platform (such as Illumina, Ion Torrent, etc.) based on experimental requirements (such as sequencing depth, throughput, cost, etc.);

[0067] Sequencing depth and coverage evaluation: Based on the complexity of the cyanobacterial community and the expected analysis accuracy, simulated data or known community structure data are used to evaluate the coverage changes at different sequencing depths and determine the optimal sequencing depth;

[0068] Library construction: 16S RNA gene was amplified from cyanobacteria samples using optimized PCR amplification conditions. Subsequently, the amplified products were purified, quantified, end-repaired, A-tailed, connected to sequencing adapters, and size screened to construct a high-quality sequencing library.

[0069] 3. Efficient and automated bioinformatics analysis:

[0070] Data quality control: Use tools such as FastQC and Trim Galore to assess and preprocess the raw sequencing data to remove contamination such as low-quality sequences, sequencing adapters, and primer sequences;

[0071] Sequence de-redundancy and clustering: CD-HIT, DADA2 and other tools are used to de-redundant the sequences after quality control, and OTU (Operational Taxonomic Units) clustering is performed based on sequence similarity to reduce data redundancy and improve analysis efficiency;

[0072] Species annotation and classification: Compare the clustered OTU representative sequences with authoritative databases such as SILVA and Greengenes, and use tools such as BLAST and RDP Classifier to perform species annotation and classification;

[0073] Community structure analysis: Bioinformatics software such as QIIME 2 and Mothur were used to calculate Alpha diversity index (such as Shannon index, Simpson index, etc.) and Beta diversity distance (such as Bray-Curtis distance, UniFrac distance, etc.) to quantitatively evaluate and compare the cyanobacterial community structure;

[0074] 4. Standardized result output and report generation:

[0075] Standardized result output: According to the analysis requirements, the species annotation results, community structure analysis results and diversity index are output in a standardized format to ensure the comparability and repeatability of the results;

[0076] Report generation: Use programming languages ​​such as R and Python, combined with graphical tools (such as ggplot2, seaborn, etc.) to generate detailed technical reports or scientific papers containing analysis results, charts and discussions.

[0077] In this embodiment, preferably, the 16srna gene of the cyanobacteria sample is amplified using optimized PCR conditions, and a high-quality sequencing library is constructed through purification, quantification, and end repair. The implementation method is as follows:

[0078] Design specific primers:

[0079] According to the sequence of the 16S RNA gene of cyanobacteria, highly specific primer pairs are designed; these primers should have an appropriate length (usually 20 bases), GC content (between 40% and 70%), and there should be no palindromic sequences inside the primers;

[0080] Optimize PCR reaction system:

[0081] Template DNA: Ensure that the DNA template of the cyanobacteria sample is of high purity and appropriate concentration (usually between 50ng and 200ng);

[0082] Primer concentration: Determine the optimal primer concentration through experiments to avoid nonspecific amplification or low amplification efficiency caused by too high or too low primer concentration;

[0083] dNTPs: The concentration of dNTPs in the reaction system should reach 100μM~200μM;

[0084] Mg^2+: As a cofactor of Taq enzyme, its concentration should be optimized to 2.0mM to ensure enzyme activity and reaction specificity;

[0085] Taq enzyme: Use high-quality Taq DNA polymerase to ensure its stability and activity at high temperatures;

[0086] Optimize PCR reaction conditions:

[0087] Denaturation temperature: usually between 93 and 95°C to ensure that the template DNA is fully denatured;

[0088] Annealing temperature: determined according to the Tm value of the primer, generally at 55°C, and the optimal annealing temperature is found by gradient PCR;

[0089] Extension temperature: The optimal reaction temperature of Taq enzyme is usually between 70 and 72°C;

[0090] Number of cycles: generally 30 to 40 cycles, adjusted according to amplification efficiency;

[0091] 2. PCR Product Purification

[0092] Electrophoresis detection: First, the size and purity of the PCR product were detected by agarose gel electrophoresis;

[0093] Purification method:

[0094] You can use commercial DNA purification kits, such as QIAquick PCR Purification Kit, or use traditional phenol-chloroform extraction and ethanol precipitation methods to remove primers, dNTPs, enzymes and other impurities in PCR products.

[0095] 3. Quantification and end repair

[0096] Quantification:

[0097] Use a spectrophotometer (such as NanoDrop) or a fluorescence quantitative PCR instrument to accurately quantify the purified PCR product;

[0098] End repair:

[0099] Due to the possible end damage during PCR, end repair is required to ensure the quality of the sequencing library;

[0100] End smoothing and phosphorylation can be performed using T4 DNA polymerase and T4 polynucleotide kinase;

[0101] 4. Construction of Sequencing Library

[0102] Ligate sequencing adapters:

[0103] Connect the repaired PCR product to a sequencing adapter (such as a sequencing adapter of the Illumina platform) to form a sequencing library;

[0104] Library screening and quality control:

[0105] Remove unligated adapters and DNA fragments that are too small by agarose gel electrophoresis or magnetic bead sorting;

[0106] Perform quality control on the library to ensure that the concentration, purity and fragment size distribution of the library meet the sequencing requirements;

[0107] Sequencing on the machine:

[0108] Load the high-quality sequencing library onto a sequencer (such as Illumina MiSeq, HiSeq, etc.) for high-throughput sequencing.

[0109] Example 2

[0110] See also Figure 1 , which is the second embodiment of the present invention, and provides a 16S RNA cyanobacteria high-throughput sequencing system, comprising

[0111] Specific amplification primer design module:

[0112] Database interface: connect to NCBI and SILVA databases to mine cyanobacteria 16srna gene sequences;

[0113] Primer design software: integrates Primer3 and Oligo software to automatically design and screen specific amplification primers;

[0114] Experimental verification platform: supports in vitro PCR amplification experiments to verify primer specificity and efficiency;

[0115] Optimize sequencing strategy and library construction modules:

[0116] Sequencing platform selection tool: recommend the optimal sequencing platform based on experimental requirements;

[0117] Sequencing depth assessment model: Evaluate the optimal sequencing depth based on simulation or known data;

[0118] Library construction automated workstation: automated equipment that integrates PCR amplification, purification, quantification, and library construction steps;

[0119] Efficient and automated bioinformatics analysis module:

[0120] Data quality control software: Integrates FastQC and Trim Galore tools to automatically remove low-quality sequences and contamination;

[0121] Sequence processing and analysis platform: supports CD-HIT, DADA2 de-redundancy, BLAST, RDP Classifier species annotation, QIIME 2, Mothur community structure analysis;

[0122] Result visualization tools: Integrate ggplot2 and seaborn graphical tools to generate intuitive analysis charts;

[0123] Standardized result output and report generation module:

[0124] Standardized result output module: automatically generates analysis results in a standardized format;

[0125] Report Generator: A combination of programming language and templates to quickly generate technical reports or scientific papers containing analysis results, charts and discussions.

[0126] Although the embodiments of the present invention have been shown and described, as detailed above, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A 16S RNA high-throughput sequencing method for cyanobacteria, characterized in that: include Specific amplification primer design and verification: Sequence database mining: Using the NCBI and SILVA databases, the BLAST tool was used to conduct extensive comparison and mining of cyanobacterial 16sRNA gene sequences to identify conservative and cyanobacterial-specific sequence regions; Primer design: Based on the mining results, Primer3 and Oligo primer design software were used to design multiple pairs of candidate amplification primers; Primer validation: Through in vitro PCR amplification experiments, the specificity, sensitivity and product quality of candidate primers are evaluated to screen out the optimal primer pair; Optimize sequencing strategy and library construction: Sequencing platform selection: Select a high-throughput sequencing platform based on experimental requirements; Sequencing depth and coverage assessment: Use simulated data or known community data to assess the optimal sequencing depth; Library construction: The 16S RNA gene of the cyanobacteria sample was amplified using optimized PCR conditions, and a high-quality sequencing library was constructed after purification, quantification, and end repair; Efficient and automated bioinformatics analysis: Data quality control: FastQC and Trim Galore tools were used to remove low-quality sequences and contamination; Sequence de-redundancy and clustering: CD-HIT and DADA2 tools were used to de-redundant and cluster into OTUs; Species annotation and classification: compare the OTU representative sequences with the authoritative database for species annotation; Community structure analysis: QIIME 2 and Mothur software were used to calculate diversity index and distance matrix and analyze community structure; Standardized result output and report generation: Standardized result output: Output the analysis results in a standardized format to ensure comparability; Report Generation: Generate detailed reports using programming languages ​​and graphical tools.

2. A 16S RNA high-throughput sequencing method for cyanobacteria according to claim 1, characterized in that: Experimental requirements include sequencing depth, throughput, and cost.

3. A 16S RNA high-throughput sequencing method for cyanobacteria according to claim 1, characterized in that: The high-throughput sequencing platforms include Illumina and Ion Torrent.

4. The method for high-throughput sequencing of 16S RNA cyanobacteria according to claim 1, characterized in that: In designing multiple pairs of candidate amplification primers, the primer design takes into account factors such as melting temperature, GC content, secondary structure formation potential, and complementarity between primers.

5. The method for high-throughput sequencing of 16S RNA cyanobacteria according to claim 1, characterized in that: The OTU representative sequences were compared with authoritative databases, including SILVA and Greengenes.

6. A 16S RNA high-throughput sequencing method for cyanobacteria according to claim 1, characterized in that: The diversity index includes Shannon index and Simpson index.

7. The method for high-throughput sequencing of 16S RNA cyanobacteria according to claim 1, characterized in that: Use programming languages ​​and graphical tools to generate detailed reports. Use R language and Python programming language, combined with graphical tools ggplot2 and seaborn, to generate detailed technical reports or scientific papers containing analysis results, charts and discussions.

8. The method for high-throughput sequencing of 16S RNA cyanobacteria according to claim 1, characterized in that: Optimized PCR conditions included denaturation temperature, annealing temperature, extension temperature, and number of cycles.

9. A 16S RNA cyanobacteria high-throughput sequencing system, characterized in that: include Specific amplification primer design module: Database interface: connect to NCBI and SILVA databases to mine cyanobacteria 16srna gene sequences; Primer design software: integrates Primer3 and Oligo software to automatically design and screen specific amplification primers; Experimental verification platform: supports in vitro PCR amplification experiments to verify primer specificity and efficiency; Optimize sequencing strategy and library construction modules: Sequencing platform selection tool: recommend the optimal sequencing platform based on experimental requirements; Sequencing depth assessment model: Evaluate the optimal sequencing depth based on simulation or known data; Library construction automated workstation: automated equipment that integrates PCR amplification, purification, quantification, and library construction steps; Efficient and automated bioinformatics analysis module: Data quality control software: Integrates FastQC and Trim Galore tools to automatically remove low-quality sequences and contamination; Sequence processing and analysis platform: supports CD-HIT, DADA2 de-redundancy, BLAST, RDP Classifier species annotation, QIIME 2, Mothur community structure analysis; Result visualization tools: Integrate ggplot2 and seaborn graphical tools to generate intuitive analysis charts; Standardized result output and report generation module: Standardized result output module: automatically generates analysis results in a standardized format; Report Generator: A combination of programming language and templates to quickly generate technical reports or scientific papers containing analysis results, charts and discussions.

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