Internal reference composition, kit and method for detecting absolute content of microorganisms based on high-throughput sequencing

Abstract

The invention discloses an internal reference composition, a kit and a method for detecting the absolute content of microorganisms based on high-throughput sequencing, the internal reference composition comprises quantitative internal references P1-P12 and an indication internal reference P0, and the sequences of the quantitative internal references P1-P12 are shown as SEQ ID NO: 1-12; the sequence for indicating the internal reference P0 is as shown in SEQ ID NO: 13. The method comprises the following steps: adding an internal reference composition consisting of internal references with different concentration gradients into a detection sample for amplification together, carrying out gel leakage detection on amplicons by adopting agarose gel, carrying out gel electrophoresis analysis on a gel chart by using software, carrying out concentration comparison on PCR products, pre-estimating the internal reference proportion according to the concentration of a gel leakage strip, and determining the content of the internal reference in the PCR products. According to the method, samples with the internal reference pre-estimation proportion being 8%-50% are conveniently selected, the method is wide in application range, low in cost and high in accuracy, different types of samples can be covered, and the applicable sample concentration range is 1 ng / mu L-50 ng / mu L.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and sequencing omics analysis, and specifically relates to an internal reference composition, a kit and a method for detecting the absolute content of microorganisms based on high-throughput sequencing. Background Art

[0002] The study of microbial populations is of great significance for understanding ecosystem functions, human health, and disease treatment. The absolute abundance of a microbial community refers to the actual number of microorganisms in a specific environment, and this data is crucial for evaluating the role and impact of microorganisms in an ecosystem. However, traditional microbial community research mainly relies on the determination of relative abundance, which cannot directly reflect the actual number of microorganisms, nor can it accurately compare the differences in microbial communities between different samples or under different conditions.

[0003] High-throughput sequencing technology randomly fragments DNA, adds adapters, prepares sequencing libraries, and then extends tens of thousands of clones in the library to detect corresponding signals and finally obtain sequence information. It can generate a large amount of sequencing data in a short time. Compared with traditional first-generation sequencing technology, high-throughput sequencing technology has higher throughput and lower cost, providing a powerful tool for in-depth research on microbial communities.

[0004] With the development of high-throughput sequencing technology (Next Generation Sequencing, NGS), especially the application of the Illumina platform, it has become possible to quantify the absolute abundance of microbial communities. Absolute quantitative sequencing adds a spike-in internal reference sequence with a known copy number to the conventional amplicon and amplifies it together with the extracted sample DNA, and then builds a library for sequencing. After the sequencing data is downloaded, the spike-in internal reference sequence added is split, and a standard curve is fitted according to its copy number and the number of sequences obtained by sequencing. The standard curve is then used to correct the number of sequences in the sample, thereby obtaining information on the type and absolute number of microorganisms.

[0005] In practical applications, the key is to select appropriate spike-in internal reference sequences, which need to be sufficiently different from the target sequence to avoid cross-reactions, while ensuring that they have similar biochemical properties to the target sequence during amplification and sequencing. In this way, the amplification efficiency and sequencing coverage of the internal reference sequence can serve as a reliable reference for the target sequence.

[0006] Currently, adding spike-in internal references with known copy numbers to samples is already a common quantitative method. By using internal references, researchers can more accurately assess the actual abundance of microorganisms in samples, which is crucial for understanding the structure and function of microbial communities and their role in ecosystems. In addition, this method also helps to eliminate variability in sample preparation and sequencing, making data obtained under different experimental conditions or at different time points more comparable.

[0007] However, adding spike-in sequences and amplifying and sequencing together with samples will cause the spike-in sequences to occupy a portion of the sequencing data volume, that is, the sequencing data of the real microorganisms in the sample will be "swallowed" by the spike-in sequences. If the proportion occupied by the spike-in sequences cannot be controlled, it will not be possible to ensure that the sample has sufficient effective data volume. On the one hand, if the amount of spike-in added is too small and the spike-in sequence is too low, it will not be sufficiently representative. In addition, when the amount of spike-in added is too small, the low concentration of the internal reference cannot be detected, and it will not be possible to fit the standard curve according to the internal reference of different concentration gradients, resulting in the failure of the quantitative experiment. On the other hand, if the amount of spike-in added is too high, the sequencing data of the real microorganisms in the sample will be diluted by the internal reference sequence, and the amount of sequencing data of the real microorganisms in the sample will be reduced, resulting in the failure to meet the expected experimental requirements. Therefore, the amount of spike-in internal reference added is crucial to the success of the experiment.

[0008] The existing patent CN 109943654 A "Method for detecting bacterial flora composition and absolute content based on internal reference sequence" discloses a quantitative method for controlling the number of spike-in sequences: after the added spike-in is amplified together with the sample, the capillary electrophoresis results are displayed by PeakScan software. Since the ratio of the absolute value of the copy number of the added "indicator internal reference" and the "quantitative reference sequence" is known, the ratio of the "quantitative reference sequence" to the total number of sequenced sequences can be obtained according to the area ratio of the indicator peak containing the indicator internal reference and the target peak containing the sample DNA and the "quantitative reference sequence". Using its ratio as a standard for judging whether the sample is sent for testing can avoid the problem of too high or too low ratio of spike-in sequences to sequencing data. However, capillary electrophoresis also has limitations, mainly high cost and low throughput. Therefore, it is very necessary to develop a high-throughput detection method. Summary of the invention

[0009] In order to solve at least one of the above problems, the present invention provides an internal reference composition, a kit and a method for detecting the absolute content of microorganisms based on high-throughput sequencing.

[0010] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides an internal reference composition for detecting the absolute content of microorganisms based on high-throughput sequencing, the internal reference composition comprising quantitative internal references P1-P12 and an indicating internal reference P0, the sequences of the quantitative internal references P1-P12 are shown in SEQ ID NO: 1-12; the sequence of the indicating internal reference P0 is shown in SEQ ID NO: 13.

[0011] The internal reference sequence P0 is used to estimate the ratio of the quantitative reference sequence to the sample sequence to be sequenced.

[0012] In some embodiments of the present invention, the quantitative internal references P1-P12 and the indicator internal reference P0 both contain binding sites for two pairs of universal primers, wherein one pair of universal primers is a forward primer 338f and a reverse primer 806R for amplifying the 16S-V3V4 fragment, and their sequences are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively; and the other pair of universal primers is a forward primer ITS-3F and a reverse primer ITS-4R for amplifying the ITS2-1 fragment, and their sequences are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0013] In some embodiments of the present invention, the quantitative internal references P1-P12 also include random sequences 1-5, and the random sequences 1-5 are respectively selected from different ones of SEQ ID NOs: 18-22; the indicator internal references also include random series 6-10, and the random series 6-10 are respectively selected from different ones of SEQ ID NOs: 23-27.

[0014] In some embodiments of the present invention, the quantitative internal references include: random sequence 1 + primer 338F + random sequence 2 + primer 806R + random sequence 3 + primer ITS-3F + random sequence 4 + primer ITS-4R + random series 5; Indicator internal reference: random sequence 6 + primer 338F + random sequence 7 + primer ITS-3F + random sequence 8 + primer 806R + random sequence 9 + primer ITS-4R + random series 10; Among them, the sequence length (bp) of the random primers satisfies the following: random sequence 1 + random sequence 2 + random sequence 3 + random sequence 4 + random sequence 5 = random sequence 6 + random sequence 7 + random sequence 8 + random sequence 9 + random sequence 10; Random sequence 2 = (random sequence 7 + primer ITS-3F + random sequence 8) - 150; Random sequence 4=(random sequence 8+primer 806R+random sequence 9)-150.

[0015] In some embodiments of the present invention, the quantitative internal references P1-P12 and the indicating internal reference P0 are mixed into a quantitative standard according to a mass ratio of P1:P2:P3:P4:P5:P6:P7:P8:P9:P10:P11:P12:P0=1:1:1:1:10:10:10:10:100:100:1000:1000:2244.

[0016] In some embodiments of the present invention, the quantitative standard is recorded as S1, which is diluted by 10 times, 100 times, 1000 times, 10000 times, and 100000 times to form quantitative standards S2-S6. The total final concentration of the quantitative reference sequence in the six quantitative standards S1-S6 is known, which is 2.52×10 9 copies / μL, 2.52×10 8 copies / μL, 2.52×10 7 copies / μL, 2.52×10 6 copies / μL, 2.52×10 5 copies / μL, 2.52×10 4 copies / μL.

[0017] The second aspect of the present invention provides a kit for detecting absolute content of microorganisms based on high-throughput sequencing, wherein the kit comprises the internal reference composition described in the first aspect.

[0018] The present invention also provides a method for using the kit of the second aspect: quantitative internal references P1-P12 and indicator internal reference P0 are mixed into a quantitative standard according to a mass ratio of P1:P2:P3:P4:P5:P6:P7:P8:P9:P10:P11:P12:P0=1:1:1:1:10:10:10:10:100:100:1000:1000:2244, which is recorded as S1, and the quantitative standard is diluted into quantitative standards S2-S6 by 10 times, 100 times, 1000 times, 10000 times, and 100000 times; the total final concentrations of the quantitative reference sequences in the six quantitative standards S1-S6 are known, which are 2.52×10 9 copies / μL, 2.52×10 8 copies / μL, 2.52×10 7 copies / μL, 2.52×10 6 copies / μL, 2.52×10 5 copies / μL, 2.52×10 4 The method further comprises mixing the sample DNA to be tested with the quantitative standard substance and amplifying the mixture; and further comprises a subsequent detection step of the amplicon after amplification.

[0019] The third aspect of the present invention provides a method for detecting the absolute content of a microbial population based on high-throughput sequencing, comprising the following steps: S1. Extract the total DNA of the sample to be tested; S2. diluting the quantitative standard substance described in claim 4 by an integer multiple of 10 to obtain quantitative standards with different total final concentrations of internal reference sequences, mixing the sample DNA to be tested with the quantitative standard substance and amplifying the mixture to obtain a 16S / ITS amplicon containing a quantitative standard substance product; S3, run the amplicon obtained in S2 by gel electrophoresis, analyze the bands by gel electrophoresis, estimate the proportion of quantitative internal reference reads according to the band concentration, and select amplicon samples with a quantitative internal reference proportion in the range of 8% to 50% for testing; S4, construct a library for the amplicon samples screened in S3 and perform sequencing, split the quantitative standard sequence according to the offline data, count the sequence numbers of the quantitative internal reference sequences P1-P12, and fit the standard curve according to the copy numbers of P1-P12; S5. Filter the internal reference sequences P1-P12 from the offline data to obtain high-quality non-internal reference valid sequences, cluster the valid sequences according to consistency, and output the quantitative results of each bacterial community in the sample.

[0020] In some embodiments of the present invention, the input amount of the indicator internal reference P0 in the quantitative standard is equal to the sum of the added amounts of the quantitative internal references P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, and P12; gel electrophoresis analysis is performed using gene tools to obtain the concentration of each band, and the sequence proportion of the quantitative internal reference is estimated based on the band concentration.

[0021] In some embodiments of the present invention, for amplicon samples whose estimated quantitative internal reference ratio is outside the range of 8% to 50%, the quantitative standard mixed with the sample is further adjusted and then re-amplified.

[0022] In some embodiments of the present invention, step S3 further includes a gel recovery step: for samples in which the quantitative internal reference sequence is estimated to account for 8% to 50%, gel recovery is performed by cutting off the strips of the quantitative internal reference and sample positions.

[0023] The length of the quantitative internal reference sequence is equal to that of the sample. On the gel, the position of the quantitative internal reference band coincides with the position of the sample band. The indicator internal reference sequence is less than the quantitative internal reference. On the gel, the indicator internal reference, sample, and quantitative internal reference can be distinguished based on the band position.

[0024] In some embodiments of the present invention, step S4 further includes a copy number correction step: the copy number of the sample flora is further corrected according to the constructed standard curve, and the absolute copy number of each flora in the sample is calculated.

[0025] In some embodiments of the present invention, step S5 also includes a step of performing quality control on the offline data, including removing adapter sequences, low-quality and repetitive sequences, to obtain high-quality sequencing data.

[0026] In an optional embodiment of the present invention, the sample to be tested is any one of soil, water, wine lees, wine mash, cellar mud, and feces. Sample DNA extraction adopts an extraction method known in the industry, and a suitable DNA extraction method is selected according to different types of samples to increase the DNA concentration and total amount; In some embodiments of the present invention, the quality control qualified offline data is filtered, and the internal reference sequences P1-P12 are obtained to obtain high-quality non-internal reference valid sequences, wherein at least 97% consistency is clustered into one OTU, and the number of OTU sequences of the sample is corrected with a standard curve to obtain the quantitative results of each bacterial community in the sample. High-quality non-internal reference valid sequences are used for OTU abundance analysis, OTU clustering analysis, species annotation analysis, and statistical analysis.

[0027] Beneficial Effects of the Invention Compared with the prior art, the present invention has the following beneficial effects: The present invention adds internal references of different concentration gradients to the test sample for amplification, uses agarose gel for gel running detection, and uses GeneTools analysis software to perform gel electrophoresis analysis on the gel image. After comparing the concentrations of PCR products, the proportion of the internal reference is estimated according to the concentration of the running gel bands, so that samples with an estimated internal reference proportion of 8%-50% can be selected conveniently.

[0028] The quantitative internal reference is set with 12 plasmids, of which 4 plasmids are set in the low concentration gradient and the lower concentration gradient respectively. This can effectively avoid the situation where the high concentration gradient plasmid and sample sequences cover the low concentration gradient and the lower concentration gradient plasmid sequences during sequencing, resulting in the inability to fit the standard curve or the incomplete standard curve and unable to quantify.

[0029] By adding internal reference DNA of different concentration gradients to environmental samples, the absolute content of different microbial communities can be quantified; during the PCR reaction, the internal reference DNA molecules will produce amplicons of expected sizes. Combining the copy number of the internal reference DNA and the number of reads output by sequencing, a standard curve between the internal reference DNA copy number (log copy number) and reads (log reads) is established, and the absolute abundance of the copy number of each bacterial community in the sample can be further calculated, which can accurately estimate the absolute abundance of species in a unit sample; this internal reference addition method has good repeatability and stability, and can truly reflect the absolute abundance of each species in the sample.

[0030] This method has a wide range of applications, low cost, high accuracy, can cover different types of samples, and is applicable to sample concentrations ranging from 1ng / μL to 50ng / μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the gradient dilution of the internal reference plasmid DNA is shown; Figure 2 A flow chart of absolute quantitative detection of microorganisms is shown; Figure 3 The top 10 absolute copy numbers at the genus level when different template DNA addition amounts and different internal reference concentration gradients of real sample 1 in implementation case 3 are shown; the horizontal axis is the amplicon that meets the internal reference ratio of 8-50%, and the vertical axis is the top 10 absolute copy numbers at the genus level of the sample; Figure 4 The top 10 absolute copy numbers at the genus level when different template DNA addition amounts and different internal reference concentration gradients of real sample 2 in implementation case 3 are shown; the horizontal axis is the amplicon that meets the internal reference ratio of 8-50%, and the vertical axis is the top 10 absolute copy numbers at the sample genus level; Figure 5 The top 10 absolute copy numbers at the genus level when different template DNA addition amounts and different internal reference concentration gradients are shown in implementation case 3; the horizontal axis is the amplicon that meets the internal reference ratio of 8-50%, and the vertical axis is the top 10 absolute copy numbers at the genus level of the sample; Figure 6 The top 10 absolute copy numbers at the genus level when different template DNA addition amounts and different internal reference concentration gradients of real sample 4 in implementation case 3 are shown; the horizontal axis is the amplicon that meets the internal reference ratio of 8-50%, and the vertical axis is the top 10 absolute copy numbers at the genus level of the sample; Figure 7 The top 10 absolute copy numbers at the genus level when different template DNA addition amounts and different internal reference concentration gradients are shown for real sample 5 in implementation case 3; the horizontal axis is the amplicon that meets the internal reference ratio of 8-50%, and the vertical axis is the top 10 absolute copy numbers at the genus level of the sample; Figure 8 The top 10 absolute copy numbers of the genus level for real sample 7 in implementation case 3 with different template DNA addition amounts and different internal reference concentration gradients are shown; the horizontal axis represents the amplicons with an internal reference ratio of 8-50%, and the vertical axis represents the top 10 absolute copy numbers of the sample genus level. DETAILED DESCRIPTION

[0032] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0034] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0035] Example 1 Design and synthesis of internal reference sequences The bacterial 16S-V3V4 fragment and the fungal ITS2-1 universal primer were inserted into the random sequence, which is an artificially edited sequence with the same length and GC content as the target fragment. After NCBI alignment, there is no match with the known species sequences in nature.

[0036] Twelve quantitative internal references and one indicator internal reference were designed, and the sequence information is shown in Table 1. The designed quantitative internal reference and indicator internal reference sequences were inserted into the plasmid.

[0037] Table 1 Internal reference sequences

[0038] The design scheme of the quantitative internal reference is as follows: random sequence 1 + primer 338F + random sequence 2 + primer 806R + random sequence 3 + primer ITS-3F + random sequence 4 + primer ITS-4R + random series 5; The design scheme of the indicated internal reference was: random sequence 6 + primer 338F + random sequence 7 + primer ITS-3F + random sequence 8 + primer 806R + random sequence 9 + primer ITS-4R + random series 10; Among them, the sequence length (bp) of the random primers satisfies the following: random sequence 1 + random sequence 2 + random sequence 3 + random sequence 4 + random sequence 5 = random sequence 6 + random sequence 7 + random sequence 8 + random sequence 9 + random sequence 10; Random sequence 2 = (random sequence 7 + primer ITS-3F + random sequence 8) - 150; Random sequence 4 = (random sequence 8 + primer 806R + random sequence 9) - 150; The random sequences 1-5 in the quantitative internal reference are selected from different ones of SEQ ID NO: 18 to SEQ ID NO: 22; the random primers 6-10 in the indicator internal reference are selected from different ones of SEQ ID NO: 23 to SEQ ID NO: 27.

[0039] In order to separate the plasmids when unloading, two recognition sequences are set for each plasmid except P1, located at the 3' end of the universal primer. The recognition sequences of each plasmid are shown in Table 2 below.

[0040] Table 2: Plasmid recognition sequences

[0041] After the synthesized plasmids were expanded and cultured, they were extracted and the qubit concentration of each plasmid was measured. Then, the mixture was mixed in the ratio of P1: P2: P3: P4: P5: P6: P7: P8: P9: P10: P11: P12: P0 = 1: 1: 1: 1: 10: 10: 10: 10: 100: 100: 1000: 1000: 2244. The plasmid concentration was accurately determined using Qubit. According to the internal reference addition amount in Table 3, each quantitative reference sequence (plasmid) was mixed, and TE (10mM Tris-Cl, 1mM EDTA, pH 8.0) was added to 1000μL, marked as S1 (quantitative standard), and oscillated to mix. The concentration of S1 was accurately determined using Qubit, and repeated 3 times independently. The concentration of S1 should be about 22.44 ng / μL, corresponding to a total number of molecules of about 5.04×10 9 Copies / μL. If the actual measured concentration difference exceeds 15%, it needs to be re-prepared; the prepared S1 needs to be divided into 5 tubes (200μL / tube), record the time of plasmid premix, and place at -80℃.

[0042] Table 3: Premixed amount of internal reference plasmid DNA

[0043] The S1 quantitative standard was diluted by 10 times, 100 times, 1000 times, 10000 times, and 100000 times to form quantitative standards S2-S6. The schematic diagram of the gradient dilution of the internal reference plasmid DNA is shown in Figure 1As shown. Experimental operations must be strictly regulated during gradient dilution of plasmid DNA. The stock solution of plasmid DNA must be shaken, mixed, and centrifuged before the experiment. Samples with too low plasmid concentrations during premixing need to be diluted before premixing. Each tube of diluted plasmid DNA must be labeled with the plasmid name and dilution multiple. To ensure the stability of the plasmid, dilute with TE. During gradient dilution, first add 90μLTE to the centrifuge tube to be diluted. Insert the gun tip 2-3mm below the surface of the stock solution to absorb the plasmid DNA. Pay attention to whether the gun tip absorbs liquid and whether there are bubbles. Then insert the gun tip into the tube to be diluted below the liquid surface and slowly discharge it. Use a pipette to blow 2-3 times to ensure that there is no residual plasmid DNA in the gun tip. Discard the gun tip, cover the tube cap, shake and mix, and centrifuge before diluting the next gradient. After each gradient dilution, the tube cap needs to be covered, shaken, mixed, and centrifuged. When doing gradient dilution, first add TE to each tube to be diluted. For example, for a 10-fold dilution, add 90 μL of TE to the centrifuge tube, then add 10 μL of plasmid DNA stock solution, shake to mix, centrifuge, and then dilute 100 times. Similarly, in the centrifuge tube marked with a 100-fold dilution, add 90 μL of TE, then add 10 μL of 10-fold diluted plasmid DNA, and so on.

[0044] According to the formula provided by Kazuyoshi Koike:

[0045] Remark: ①6.02×10 23 It is an approximation of Avogadro's constant, which represents the number of particles (atoms, molecules, ions, etc.) contained in 1 mol of a substance; ②660 (Dalton) is the average molecular weight of a DNA base pair (sodium salt); ③109 is the conversion factor between g and ng.

[0046] The copy number of each plasmid at different concentration gradients was calculated, as shown in Table 4 below.

[0047] Table 4: The copy number of plasmids at different concentration gradients

[0048] Among them, S3~S5 can cover most of the samples.

[0049] Example 2 Artificial Simulation Community Experiment Twelve pure bacterial strains were selected, and the concentrations of the 12 pure bacterial strains were measured by Qubit, and the purity of the enzyme strain pure bacteria was measured by Nanodrop. Each strain was mixed into an artificial simulated community according to different input amounts. The specific information of the 12 pure bacterial strains is shown in Table 5.

[0050] Table 5 Strain quality information

[0051] After some preliminary experiments, it was found that for the above-mentioned artificial simulated communities, 12.44 ng and 6.22 ng were more suitable for the three gradients of S1, S2, and S3. When the gradient was lower, it was determined that the proportion of the internal reference was too low.

[0052] Therefore, the above artificial simulated community was diluted into three gradients: 12.44 ng / μL, 6.22 ng / μL, and 3.11 ng / μL, and the artificial simulated communities with these three gradients were mixed with six different concentration gradient internal references S1, S2, S3, S4, S5, and S6 for amplification; it was named "simulated community amplification input amount-internal reference concentration gradient", such as 12.44-S1 means that the simulated community amplification input amount is 12.44 ng, and the internal reference concentration gradient is S1.

[0053] Amplification was performed using 16s-v3v4 primers. The amplification system was 25 μL, of which 1 μL was input into the artificial simulated community, 1 μL was input into the internal reference plasmid, and the system was supplemented with water.

[0054] After amplification, 1% agarose gel was used for gel running and the gel image was saved. The saved gel image was analyzed by gel electrophoresis using GeneTools analysissoftware (Version4.03.05.0 SynGene). After comparing the concentrations of PCR products, the proportion of internal reference was estimated based on the concentration of the gel bands.

[0055] The estimated proportion of internal references was calculated based on the gel map, and samples with an estimated proportion of internal references between 8% and 50% were selected for subsequent experiments. There were a total of 4 samples, namely 12.44-S3, 6.22-S3, 3.11-S3, and 3.11-S4.

[0056] According to the concentration of PCR product bands obtained by GeneTools analysis software (Version4.03.05.0 SynGene), the required mixed sample volume of each sample was calculated according to the equal amount principle, and each PCR product was mixed and quantified by Qubit. The PCR mixed product was recovered using the HiPure Gel Pure DNA Mini Kit gel recovery kit, the target DNA fragment was eluted and recovered by TE buffer, and the concentration of the recovered DNA was measured. Finally, the ALFA-SEQ DNA Library PrepKit library construction kit was used for library construction, and the index code for distinguishing samples and the universal sequence required by the sequencing platform were added to both ends of the library; and the constructed amplicon library was sequenced using the Illumina platform. After sequencing, the samples were split according to the sample barcode code, and the data was quality controlled, low-quality amplicon sequences were removed, and similar sequences were clustered. Generally, sequences with a similarity greater than or equal to 97% were divided into one OTU, which was divided into a small number of taxonomic units, and species annotation was performed based on these taxonomic units.

[0057] The standard curve was fitted based on the internal reference concentration and the number of sequences. A linear regression equation between the number of gradient plasmid DNA copies (log copies) and the number of sequences (log reads) was established based on the plasmid copy number information and the number of internal reference DNA reads in the sequencing results, where y is the logarithm of the copy number and x is the logarithm of the reads. At the same time, the actual proportion of the internal reference was counted. The above experimental results are shown in Table 6.

[0058] Table 6 Estimated and actual proportions of internal reference and standard curve

[0059] The results show that estimating the proportion of internal references in the offline data by gel electrophoresis analysis using GeneTools analysis software can more accurately reflect the proportion of internal references in the sequencing data.

[0060] The copy number of the sample flora was further corrected by fitting the standard curve, and the absolute copy number of each flora in the sample was calculated, as shown in Table 7. The reads corresponding to the sample were substituted into the linear regression formula to calculate the log10 (copy number) of the sample, recorded as c:

[0061] Get the number of copies d (copies) of the sample nucleic acid:

[0062] According to the total amount of template DNA added to amplification n (unit: ng), the number of nucleic acid copies (copies / ng) is obtained; The number of copies of nucleic acid: the unit is (copies / ng nucleic acid):

[0063] Table 7 Sequence numbers of samples and absolute copy numbers after calibration by standard curve

[0064] By comparing the species copy numbers measured by adding internal references of different concentration gradients and different sample input amounts in the same artificial simulated community, it was found that the copy numbers of species in the same artificial simulated community were basically the same, indicating that there was no obvious difference in the absolute quantification of bacteria in the samples by adding internal references of different concentrations and different sample input amounts. That is, the internal reference method has good repeatability and stability, and can truly reflect the absolute abundance of each species in the sample.

[0065] From the above experimental results, it can be seen that the S1 and S2 internal reference concentration gradients are not suitable for amplification with samples, otherwise the proportion of internal reference sequences is likely to be too high, and the S6 internal reference concentration gradient is easily indicated by the internal reference without target bands after amplification and running the detection gel with the sample, especially when the quality of the artificial simulated community strain is high, so the proportion of internal reference will be 0.

[0066] Example 3 Real sample detection Four different types of microorganisms from real environmental samples were selected from the sample library for absolute quantitative experiments. The absolute quantitative detection flow chart of microorganisms is shown in the figure. Figure 1 shown.

[0067] Among the real samples, three samples are soil samples, three are Daqu samples, three are cotton swab samples, and one is a water body sediment sample. The quality of the four sample types is different. Different concentration gradient internal references are added according to the sample input amount. The sample input amount is divided into three gradients of 1ng, 10ng, and 50ng. When the input is 1ng, 1μL S4 and S5 are added. When the input is 10ng and 50ng, 1μL S4 and S5 are added. Different samples, different sample input amounts, and different concentration gradient plasmids together constitute 60 amplification samples, named "sample number-internal reference concentration gradient-sample input amount", such as: "1-S4-1" means that the sample number is 1, the internal reference concentration gradient is S4, and the sample input amount is 1ng. The specific quality of the sample is shown in Table 8 below.

[0068] Table 8 Real sample quality

[0069] The amplification program was 94℃ 5min, 94℃ 30s, 53℃ 30s, 72℃ 30s, 72℃, 8min, 4℃ ∞. Amplification was performed according to this amplification program and the samples were temporarily stored at 4℃ after the amplification. The program was stopped and the machine was turned off. The PCR products were then taken out and run on gel. The concentration of PCR product bands was obtained using GeneTools analysis software (Version4.03.05.0 SynGene). After comparing the concentrations of PCR products, the proportion of internal reference was estimated based on the concentration of the running gel bands. Samples with an estimated internal reference proportion of 8% to 50% were selected for subsequent experiments. A total of 18 amplicons had an estimated internal reference proportion of 8% to 50%, and the internal reference proportions of the remaining amplicons were too high or too low.

[0070] The required mixed sample volume for each sample was calculated according to the equal amount principle, and each PCR product was mixed and then quantified by Qubit. The PCR mixed product was recovered using the HiPure Gel Pure DNA Mini Kit gel recovery kit, the target DNA fragment was eluted and recovered by TE buffer, and the concentration of the recovered DNA was measured. Finally, the ALFA-SEQ DNA Library Prep Kit was used to build the library, and the index code used to distinguish the samples and the universal sequence required by the sequencing platform were added to both ends of the library; and the constructed amplicon library was sequenced using the Illumina platform. After sequencing, the samples were split according to the sample barcode code, and the data was quality controlled, low-quality amplicon sequences were removed, the internal reference sequences were split, and the proportion of the internal reference sequences in the sequencing data was calculated. The results are shown in Table 9 below.

[0071] Table 9 Estimated and actual proportions of internal reference and standard curve data

[0072] Judging from the sequencing results, due to sample quality issues, the amplicons of cotton swab samples 8, 9, and 10 did not reach the internal reference ratio of 8% to 50%. That is, there is a greater risk in conducting absolute quantitative amplicon experiments with low-quality samples, and the proportion of internal references after amplification is too high. Therefore, it is not recommended to continue amplifying such samples or to choose risky amplification on the machine.

[0073] The sample amplification input amount was divided into three gradients of 1ng, 10ng, and 50ng, which were mixed with different internal reference concentration gradients for amplification. The estimated proportion of the internal reference was calculated based on the results. The results showed that the amplicons in all three gradients reached the internal reference proportion range of 8%~50%, that is, this amplification scheme is suitable for samples in the concentration range of 1ng / μL~50ng / μL.

[0074] Among the 60 amplicons in 10 samples, 18 amplicons in 6 samples that met the internal reference ratio standard were sent for testing. The samples were sequenced using high-throughput sequencing technology to obtain sample OTU clustering, and the copy number of the samples was converted based on the standard curve fitted by the internal reference sequence number and the copy number. The conversion unit is copies / ng. The absolute copy number of the top 10 at the genus level is selected for display, such as Figure 3-8 As shown, each picture represents a sample, the horizontal axis is the sample name, which is equivalent to the repetition of the same sample, and the vertical axis is the copy number of the top 10 at the genus level.

[0075] The results showed that the method of the present invention can effectively detect the absolute copy number of 16S at each classification level in soil, Daqu and water sediment samples, and has extremely high consistency between replicates, that is, the detection results of the method of the present invention have high repeatability.

[0076] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. An internal reference composition for detecting absolute content of microorganisms based on high-throughput sequencing, characterized in that: The internal reference composition includes quantitative internal references P1-P12 and an indicating internal reference P0, wherein the sequences of the quantitative internal references P1-P12 are shown in SEQ ID NOs: 1-12; and the sequence of the indicating internal reference P0 is shown in SEQ ID NO:

13.

2. The internal reference composition according to claim 1, characterized in that: The quantitative internal references P1-P12 and the indicator internal reference P0 both contain binding sites for two pairs of universal primers, one pair of universal primers being a forward primer 338f and a reverse primer 806R for amplifying the 16S-V3V4 fragment, and their sequences are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively; the other pair of universal primers being a forward primer ITS-3F and a reverse primer ITS-4R for amplifying the ITS2-1 fragment, and their sequences are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

3. The internal reference composition according to claim 2, characterized in that: The quantitative internal references P1-P12 also include random sequences 1-5, and the random sequences 1-5 are respectively selected from different ones of SEQ ID NOs: 18-22; the indicator internal references also include random series 6-10, and the random series 6-10 are respectively selected from different ones of SEQ ID NOs: 23-27.

4. The internal reference composition according to any one of claims 1 to 3, characterized in that: The quantitative internal references P1-P12 and the indicating internal reference P0 are mixed into a quantitative standard according to the mass ratio of P1:P2:P3:P4:P5:P6:P7:P8:P9:P10:P11:P12:P0=1:1:1:1:10:10:10:10:100:100:1000:1000:2244.

5. A kit for detecting absolute content of microorganisms based on high-throughput sequencing, characterized in that: The kit comprises the internal reference composition according to any one of claims 1 to 4.

6. A method for detecting absolute content of microbial populations based on high-throughput sequencing, characterized in that: The steps include: S1. Extract the total DNA of the sample to be tested; S2. diluting the quantitative standard substance described in claim 4 by an integer multiple of 10 to obtain quantitative standards with different total final concentrations of internal reference sequences, mixing the sample DNA to be tested with the quantitative standard substance and amplifying the mixture to obtain a 16S / ITS amplicon containing a quantitative standard substance product; S3, run the amplicon obtained in S2 by gel electrophoresis, analyze the bands by gel electrophoresis, estimate the proportion of quantitative internal reference reads according to the band concentration, and select amplicon samples with a quantitative internal reference proportion in the range of 8% to 50% for testing; S4, construct a library for the amplicon samples screened in S3 and perform sequencing, split the quantitative standard sequence according to the offline data, count the sequence numbers of the quantitative internal reference sequences P1-P12, and fit the standard curve according to the copy numbers of P1-P12; S5. Filter the internal reference sequences P1-P12 from the offline data to obtain high-quality non-internal reference valid sequences, cluster the valid sequences according to consistency, and output the quantitative results of each bacterial community in the sample.

7. The method according to claim 6, characterized in that: Step S3 also includes a gel recovery step: for samples in which the quantitative internal reference sequence is estimated to account for 8% to 50%, gel recovery is performed by cutting off the strips at the positions of the quantitative internal reference and the sample.

8. The method according to claim 6, characterized in that: Step S5 also includes a copy number correction step: the copy number of the sample flora is further corrected according to the constructed standard curve, and the absolute copy number of each flora in the sample is calculated.

9. The method according to claim 6, characterized in that: Step S5 also includes a step of quality control of the data, including the removal of adapter sequences, low-quality and repeated sequences, to obtain high-quality sequencing data.

10. The method according to any one of claims 6 to 9, characterized in that: The sample to be tested is any one of soil, water, wine lees, wine mash, cellar mud, and feces.

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