Fungus population absolute quantification method based on Pacbio platform

Abstract

The invention develops a method for absolute quantification of fungal population based on a Pacbio three-generation long fragment sequencing platform and application thereof, and belongs to the technical field of microbial amplicon detection. According to the method, the synthesized chimeric DNA is used as an internal standard, and a fungal community in a sample is absolutely quantified on the basis of an amplicon high-throughput sequencing platform. 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 high-throughput sequencing on the ITS full-length sequence of the fungus through PCR amplification; the high-throughput sequencing results calculate the relative abundance and absolute abundance of the amplicon family (e.g., the copy number of fungal ITS per unit mass sample). 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 in particular 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 is widely used in the detection of microbial diversity.

[0003] ITS1 is located between the 18S and 5.8S of the eukaryotic ribosomal rDNA sequence, and ITS2 is located between the 5.8S and 28S of the eukaryotic ribosomal rDNA sequence. Since the ITS region is excised during ribosomal RNA processing and does not play a functional role, the selection pressure is relatively small during the evolution process, and the evolution rate is about 10 times that of 18S rDNA. It belongs to a moderately conserved region. Using it can study taxonomic ranks below the species level. Compared with the second-generation Illumina sequencing platform that can only select the variable regions of ITS1 or ITS2, the third-generation full-length sequencing can sequence the full length of ITS, thereby improving the annotation of microbial diversity from the "genus" level to the "species" level, and can more accurately describe the diversity of microbial communities.

[0004] However, the diversity analysis of relative abundance ignores the actual differences in the total microbial biomass between different samples, resulting in deviations in the analysis results; while absolute quantification analysis measures the ITS gene copy numbers of each microorganism in the sample, so as to reflect the true quantity of each microorganism in the sample and the true differences between inter-group samples. Therefore, absolute quantification analysis can better reflect the true changes in the microbial community of the sample compared with relative quantification analysis. The currently commonly used absolute quantification method 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 uses synthetic chimeric DNA as an internal standard for absolute quantification of ITS full-length amplicons. The specific method is as follows: The synthetic chimeric DNA is directly added to environmental samples; genomic DNA of the environmental samples is extracted; the full-length fungal ITS sequences are amplified by PCR and subjected to high-throughput sequencing; the relative and absolute abundances of amplicon families are calculated from the high-throughput sequencing results. The present invention uses highly complex environmental samples as the research object and proves that this method can sensitively and accurately identify the relative and absolute abundances of microbial populations. This method is suitable for simultaneous detection of multiple microbial communities, including environmental samples from soil, samples from the human gut, 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

[0006] 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.

[0007] The object of the present invention is to provide an analytical method for absolute quantification of fungi based on the Pacbio long-read sequencing platform in view of the deficiencies in the prior art.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] S1. Design and synthesize chimeric DNA. The elements included in the design are: (1) primer binding sites (PBSs) containing the full length of ITS; (2) optimized synthetic filler sequences, whose length and GC content are the same as those of the target in vivo and are not affected by the microorganisms in the environmental samples; (3) synthetic DNA sources that are easy to obtain and easy to process.

[0010] S2. Weigh a fixed amount of soil sample and add a quantitative known concentration of chimeric internal standard DNA.

[0011] S3. Extract the genomic DNA of soil microorganisms mixed with internal standard DNA in S2. Use a soil DNA extraction kit to process the samples.

[0012] S4. PCR amplify the genomic DNA of soil microorganisms extracted in S3: Use ITS1 and ITS4 primers for PCR amplification to obtain full-length ITS amplicons, and purify them.

[0013] S5. Perform high-throughput sequencing on the PCR products purified in S4, and process the sequencing data using customized Linux and Python scripts, supplemented with scripts of FASTX-toolkit (http: / / www.hannonlab.cshl.edu).

[0014] S6. Analyze the OTUs results obtained by high-throughput sequencing and calculate the relative and absolute quantification of soil microbial communities.

[0015] The present invention uses synthetic chimeric DNA as an internal standard to analyze the absolute quantification of ITS full-length amplicons. The method includes the following steps: The synthetic chimeric DNA is directly added to environmental samples; after extracting the total DNA of the samples, specific primers ITS1 and ITS4 with Barcodes are used for PCR amplification, and the products are purified, quantified, and homogenized to form a sequencing library (SMRTBell). The constructed library is first subjected to library quality inspection, and the library with qualified quality inspection is sequenced using PacBio Sequel. The data obtained from PacBio Sequel is in bam format, and CCS files are exported through the smrtlink analysis software. The data of different samples are identified according to the Barcode sequence and converted into fastq format data; the high-throughput sequencing results are compared in a database, and the relative abundances of microbial communities are obtained through the obtained taxonomic units; the absolute abundances of microbial communities are calculated according to the internal standard chimeric DNA with known concentration. This project uses highly complex environmental samples as research objects to prove that this method can sensitively and accurately identify the absolute abundances of specific populations. This method is suitable for the specific analysis of ITS full-length amplicons, including environmental samples from soil, samples from the human intestine, and samples from food samples. Brief Description of the Drawings

[0016] Figure 1 Shown is the variable region of the full length of ITS;

[0017] Figure 2 Shown is the fungal community diversity in soil samples (A. absolute abundance; B. relative abundance); Detailed Embodiments

[0018] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand the 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 embodiments, 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.

[0019] 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. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0020] Examples

[0021] S1. Collect soil samples from sandy land and meadow areas, remove weeds and litter on the ground, and sieve them for later use.

[0022] S2. Design chimeric DNA, primers containing two hypervariable region sequences in ITS, and synthesize chimeric internal standard plasmid.

[0023] S3. Weigh 300 mg of soil sample and add 100 μl of chimeric internal standard plasmid.

[0024] S4. Use the soil DNA extraction kit DNeasy PowerSoil Kit to extract total microbial DNA from the soil sample. The experimental steps are as follows:

[0025] (1) Add 160 μl of reagent C1 to the soil sample mixed with chimeric internal standard plasmid, and vortex for 10 min.

[0026] (2) Centrifuge at 12000 rpm for 30 s.

[0027] (3) After transferring the supernatant, add 250 μl of reagent C2, vortex to mix evenly, and place on ice for 5 min.

[0028] (4) Centrifuge at 12000 rpm for 1 min, transfer 600 μl of supernatant, then add 200 μl of reagent C3, vortex to mix evenly, and place on ice for 5 min.

[0029] (5) Centrifuge at 12000 rpm for 1 min, transfer 750 μl of supernatant, then add 1200 μl of reagent C4, and vortex to mix evenly.

[0030] (6) Add 700 μl of the solution to the MB Spin Column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0031] (7) Repeat step (6) until all the solution is centrifuged.

[0032] (8) Add 500 μl of reagent C5, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0033] (9) Spin empty at 12000 rpm for 1 min, transfer the column to a 1.5 mL centrifuge tube.

[0034] (10) Add 50 μl of reagent C6, heat at 55 °C for 10 min, centrifuge at 12000 rpm for 30 s, and collect the DNA.

[0035] S5. Use ITS1 and ITS4 with specific Barcode at the full length of ITS to perform PCR amplification on the purified DNA.

[0036]

[0037] Pool the final PCR products, and use the PCR products for purification, quantification, and normalization to form a sequencing library.

[0038] S6. After quality identification of the amplification products, perform high-throughput sequencing, and at least 50,000 effective CONNECT TAGS are produced for the DNA of each soil sample;

[0039] S7. Compare the sequencing results with the corresponding database and cluster them into operational taxonomic units (abbreviated as OTU in English);

[0040] S8. Perform bioinformatics analysis to obtain the relative abundances of the soil fungal community composition;

[0041] S9. Calculate the absolute abundances of the soil fungal community according to the relative content of the internal reference of the chimeric DNA.

[0042] The analysis results are as follows:

[0043] As Figure 2 shown in the results, the relative abundances and absolute abundances of the fungal community are revealed.

[0044] In the present invention, we designed a synthetic sequence that does not exist in the natural environment. In this way, after using PCR amplification to obtain the full-length ITS fragment and performing high-throughput sequencing, the synthetic sequence can be easily separated from the sequencing results of microorganisms, thus serving as a quantitative internal reference.

[0045] In summary, amplicon sequencing is to perform PCR amplification on 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, which poses a serious 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 on the microorganisms in the sample. Currently, it is widely used in microbial diversity detection. These specific genetic materials all include evolutionarily conserved regions and evolutionarily variable regions, which can be amplified from a small amount of DNA using PCR technology. Moreover, gene sequences have high variability even among closely related species. Therefore, amplicon sequencing has been widely used in taxonomy and molecular systematics. Compared with second-generation amplicon sequencing that can only analyze one of the variable regions, ITS1 or ITS2, the full-length ITS amplicon sequencing based on the Pacbio third-generation sequencing platform can more accurately identify the species of fungi. By simply adding synthetic internal standard DNA to the sample, full-length ITS sequencing can be obtained using the mature Pacbio platform, which can not only study the relative content of the fungal community but also analyze the absolute content of the fungal community.

Claims

1. An analytical method for absolute quantification of fungal 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 and contains the ITS1 and ITS4 primers used for ITS full-length amplification, which can easily isolate this fragment from the sequencing results of microorganisms, thus serving as a quantitative internal standard; S2. Add the designed and synthesized chimeric internal standard plasmid to the soil sample and extract the soil genomic DNA; S3. Perform PCR using the ITS full-length primers to obtain the ITS amplicon products in the sample of S2; S4. Perform high-throughput sequencing on the PCR products obtained in S3; S5. Sequentially perform sequence clarity and quality analysis on the amplicon sequencing to screen out qualified data that meet the requirements; S6. Combine and process 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. Perform species annotation on the data obtained in step S5 to obtain species classification annotation information at different taxonomic levels; S9. Perform diversity 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, wherein, There are three key factors in the designed synthetic sequence: (1) contain primer binding sites (PBSs) for the full length of ITS; (2) optimized synthetic filler sequence, the length and GC content of which are the same as the in vivo target; (3) 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 reference plasmid and the soil sample should not vary too much, which is likely to 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 universal primers for identifying species and are sequences contained in the chimeric internal standard.

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