Bacterial community absolute quantification method based on Pacbio platform

Abstract

The invention develops a bacterial population absolute quantification method based on a Pacbio three-generation long fragment sequencing platform and application thereof, and belongs to the technical field of microbial amplicon detection. The synthetic chimeric DNA is used as an internal standard, and the microbial community in the sample is absolutely quantified at the same time. The specific method comprises the following steps: directly adding the synthesized chimeric DNA into an environmental sample; extracting genome DNA of the environment sample; carrying out PCR amplification on the 16S full-length sequence and carrying out high-throughput sequencing; and calculating relative abundance and absolute abundance of the amplicon family according to a high-throughput sequencing result. A highly complex environmental sample is taken as a research object to prove that the method can sensitively and accurately quantify the absolute abundance of a specific group. The method is widely applicable to microbial community analysis of samples from different sources, including environmental samples from soil, samples from plant tissues, samples from human intestinal tracts, and samples from food samples. The method disclosed by the invention is simple to operate, accurate in detection and wide in application, and has a wide market prospect in identification and analysis of microbial communities.

Description

Technical Field

[0001] The present invention relates to the fields of microbial informatics and biotechnology, and particularly relates to a method for absolute quantification analysis of microbial populations based on high-throughput sequencing. Background Art

[0002] Amplicon sequencing is to sequence specific-length PCR products or captured fragments. Currently, it mainly uses high-throughput sequencing technology to sequence specific gene fragments in a specific environment. Most natural microorganisms cannot be isolated and cloned by traditional isolation and culture methods, which poses a severe challenge to the qualitative and quantitative analysis of natural microorganisms and the exploration of microbial diversity. Amplicon sequencing can overcome the weaknesses of traditional isolation and culture and perform qualitative and quantitative analysis of microorganisms in samples. Currently, it has been widely used in microbial diversity detection.

[0003] The 16S rRNA gene is unique to prokaryotes and has a very high copy number in prokaryotes. The full-length 1542bp DNA sequence contains 9 intervening hypervariable regions. The 16S rRNA gene sequence with both specificity and conservativeness is widely used as a microbial marker in research. Currently, the second-generation sequencing technology has become the mainstream means for microbial research, and the 16S rDNA V3-V4 region sequencing method is mainly used. However, due to the limitation of read length and the ability to only analyze to the genus level, it is impossible to analyze to the species level. The PacBio sequencing platform can complete fragments with an average read length of 3-5 kb and can easily amplify and sequence the full-length sequence of the 16S rRNA gene (including 9 variable regions). On the one hand, the analysis of the full-length 16S amplicon sequence can annotate more OTUs and significantly improve the accuracy of annotated species; on the other hand, it can improve the resolution of species annotation and achieve species annotation at the "species" level. Therefore, the 16S full-length amplicon analysis has a wider sample and species applicability and avoids the preference for different sample sources and species brought by different sequencing regions of conventional amplicon sequencing.

[0004] However, the diversity analysis of relative abundance ignores the existing real differences in the total microbial biomass between different samples, resulting in deviations in the analysis results; while absolute quantification analysis measures the copy number of 16S genes of each microorganism in a sample, thereby reflecting the true quantity of each microorganism in the sample and the real differences between inter-group samples. Therefore, absolute quantification analysis can better reflect the real changes in the microbial community of the sample compared to relative quantification analysis. Currently, the commonly used method for absolute quantification is mainly real-time fluorescence quantitative PCR. This method cannot achieve high-throughput analysis. More importantly, it is costly and has high requirements for the quality of sample genomic DNA.

[0005] The present invention innovatively synthesizes chimeric DNA, which contains 9 amplicon binding sites commonly used in the 16S variable region ( Figure 1 ), and is used as an internal standard for absolute quantification of the full-length 16S. The specific method is as follows: The synthesized chimeric DNA is directly added to the environmental sample; the genomic DNA of the environmental sample is extracted; the full-length 16S sequences of fungi and bacteria are amplified by PCR and subjected to high-throughput sequencing; the relative abundance and absolute abundance of the amplicon family are calculated from the high-throughput sequencing results (such as the prokaryotic 16S per unit mass of the sample).

[0006] The present invention uses highly complex environmental samples as the research object and proves that this method can sensitively and accurately identify the absolute abundance of the population. This method is suitable for simultaneously detecting bacterial communities, including environmental samples from soil, samples from the human intestine, and samples from food. The method of the present invention is simple to operate, accurate in detection, and widely applicable, and has broad market prospects in the identification and analysis of microbial communities. Summary of the Invention

[0007] The method and application for analyzing the absolute abundance of microbial communities based on high-throughput sequencing technology of the present invention belong to the technical field of microbial amplicon detection.

[0008] The purpose of the present invention is to provide an analytical method for absolute quantification of microorganisms based on the sequencing of the full-length bacterial 16S amplicon in view of the deficiencies in the prior art.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is:

[0010] S1. Design and synthesize chimeric DNA. The elements included in the design are: (1) containing 9 primer binding sites (PBSs) commonly used in the full-length 16S; (2) an optimized synthetic filler sequence with the same length and GC content as the target in vivo; (3) an easily obtainable and easily processable synthetic DNA source.

[0011] S2. Weigh a fixed amount of environmental sample and add a quantitatively known concentration of chimeric internal standard DNA.

[0012] S3. Extract the genomic DNA of the environmental sample containing the internal standard DNA.

[0013] S4. PCR amplify the genomic DNA extracted in S3, perform PCR amplification with specific primers to obtain the corresponding amplicons. Pool the final PCR products and purify them using a PCR purification kit.

[0014] S5. The PCR products purified in S4 are subjected to high-throughput sequencing, and the sequencing results are processed using customized Linux and Python scripts, supplemented by the scripts of FASTX-toolkit (http: / / www.hannonlab.cshl.edu).

[0015] S6. Analyze the OTUs results obtained by high-throughput sequencing to analyze the relative and absolute quantification of the microbial community in the sample.

[0016] The present invention uses synthetic chimeric DNA as an internal standard to analyze the absolute quantification of different amplicon families. It includes the following steps: adding internal standard DNA with known concentration to environmental samples; after extracting the total DNA of the samples, using the 16S full-length primers 27F and 1492R with Barcode (and other full-length primers) for PCR amplification, and purifying, quantifying, and normalizing the products to form a sequencing library (SMRT Bell). The constructed library is first subjected to library quality inspection, and the library with qualified quality inspection is sequenced using PacBio Sequel; the high-throughput sequencing results are compared in the database, and the relative abundance of the microbial community is obtained through the obtained taxonomic units; the absolute abundance of the microbial community is calculated based on the internal standard DNA. This project uses highly complex environmental samples as the research object to prove that this method can sensitively and accurately identify the absolute abundance of specific populations. This method is applicable to any amplicon-specific group, including environmental samples from soil, samples from the human intestine, and samples from food samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown are 9 variable amplification regions of the 16S full length;

[0018] Figure 2 Shown are the bacterial community diversities in soil samples (A. absolute abundance; B. relative abundance). DETAILED DESCRIPTION OF THE INVENTION

[0019] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0020] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Embodiment

[0022] S1. Collect soil samples from sandy land and meadow areas, remove ground weeds and withered branches and fallen leaves, and then sieve them for standby.

[0023] S2. Design chimeric DNA, primers containing 9 hypervariable region sequences in 16S, and synthesize chimeric internal standard plasmids.

[0024] S3. Weigh 300 mg of soil samples and add 100 μl (500 pg) of chimeric internal standard plasmids.

[0025] S4. Use the soil DNA extraction kit DNeasy PowerSoil Kit to extract the total microbial DNA in the soil samples. The experimental steps are as follows:

[0026] (1) Add 60 μl of reagent C1 to the soil samples mixed with chimeric internal standard plasmids, and vortex for 10 min.

[0027] (2) Centrifuge at 12,000 rpm for 30 s.

[0028] (3) After transferring the supernatant, add 250 μl of reagent C2, vortex and mix well, and place on ice for 5 min.

[0029] (4) Centrifuge at 12,000 rpm for 1 min. After transferring 600 μl of the supernatant, add 200 μl of reagent C3, vortex and mix well, and place on ice for 5 min.

[0030] (5) Centrifuge at 12,000 rpm for 1 min. After transferring 750 μl of the supernatant, add 1,200 μl of reagent C4, and vortex and mix well.

[0031] (6) Add 700 μl of the solution to the MB Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0032] (7) Repeat step (6) until all the solution is centrifuged.

[0033] (8) Add 500 μl of reagent C5, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid.

[0034] (9) Spin empty at 12,000 rpm for 1 min, transfer the column to a 1.5 mL centrifuge tube.

[0035] (10) Add 50 μl of reagent C6, heat at 55 °C for 10 min, centrifuge at 12,000 rpm for 30 s, and collect the DNA.

[0036] S5. Use 16S full-length 27F and 1492R containing specific Barcode to perform PCR amplification on the purified DNA.

[0037]

[0038] Pool the final PCR products, and use the PCR products for purification, quantification, and homogenization to form a sequencing library.

[0039] S6. After quality identification of the amplification products, perform high-throughput sequencing, and at least 50,000 effective CONNECT TAGS are generated for the DNA of each soil sample.

[0040] S7. Compare the sequencing results with the corresponding database and cluster them into operational taxonomic units (abbreviated as OTU in English).

[0041] S8. Perform bioinformatics analysis to obtain the relative abundances of the soil microbial community composition.

[0042] S9. Calculate and analyze the absolute abundances of the soil microbial community based on the content of the internal standard DNA.

[0043] The analysis results are as follows:

[0044] As Figure 2 shown in the results, the relative abundances and absolute abundances of the bacterial microbial community are revealed.

[0045] In the present invention, we designed an artificially synthesized sequence that does not exist in the natural environment. After performing high-throughput sequencing on the fragment obtained by PCR amplification, this artificially synthesized sequence can be easily separated from the sequencing results of microorganisms, thereby playing the role of a quantitative internal reference.

[0046] In summary, amplicon sequencing is to perform PCR amplification on the conserved fragments of microorganisms and then perform high-throughput sequencing on the PCR fragments. More than 90% of natural microorganisms cannot be isolated and purified into colonies by traditional methods. Amplicon sequencing can overcome the weaknesses of traditional isolation and culture, qualitatively and quantitatively analyze the microorganisms in the sample, and is currently widely used in microbial diversity detection. The full length of 16S rDNA can contain 9 variable regions, which can more accurately identify the species and genera of bacteria. Therefore, amplicon sequencing has been widely used in taxonomy and molecular systematics. By adding internal standard DNA to the sample, while performing high-throughput sequencing, not only the relative content of bacteria in the sample can be analyzed, but also the absolute content of bacteria can be accurately obtained.

Claims

1. An analytical method for absolute quantification of microbial populations based on high-throughput sequencing, the main steps of which include: S1. Design a synthetic sequence that does not exist in the natural environment. After high-throughput sequencing of the fragment obtained by PCR amplification, this fragment can be easily separated from the sequencing results of microorganisms, thus serving as a quantitative internal reference. S2. Mix the designed and synthesized chimeric internal standard DNA with the sample, and then extract the total DNA of the sample. S3. Amplify the total DNA in S2 using 16S full-length primers (27F and 1492R) containing Barcode to obtain PCR products. S4. Perform high-throughput sequencing on the PCR products obtained in S3. S5. Sequentially perform sequence clarity and quality analysis on the amplified sequencing to screen out qualified data that meet the requirements. S6. Combine and further screen the data obtained in step S5. S7. Denoise, de-chimera, and remove errors caused by sequencing errors or PCR amplification from the data obtained in step S5. S8. Annotate the species of the data obtained in step S5 to obtain species classification annotation information at different taxonomic levels. S9. Perform relative quantification and absolute quantification analysis on the data obtained in step S5.

2. The method for absolute quantification of microbial populations based on high-throughput sequencing according to claim 1, characterized in that, There are three key factors in the designed synthetic sequence: (1) containing 9 variable amplification region binding sites (PBSs) of 16S full length; (2) an optimized synthetic filler sequence, the length and GC content of which are the same as the in vivo target and are easy to distinguish from environmental microorganisms; (3) an easily obtainable and easy-to-process synthetic DNA source.

3. The method for absolute quantification of microbial populations based on high-throughput sequencing according to claim 1, characterized in that, The mixing ratio of the chimeric internal standard plasmid and the soil sample should not vary too much, as this can easily cause experimental errors.

4. The method for absolute quantification of microbial populations based on high-throughput sequencing according to claim 1, characterized in that, The primers in the PCR amplification are species-specific primers for identification, and are sequences contained in the chimeric internal standard DNA.

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