CN105331684A - Composite tag for high-throughput sequencing of biological diversity of fungi in environment and application of composite tag - Google Patents

Abstract

The invention discloses a composite tag for high-throughput sequencing of environmental fungal biodiversity and its application. The sequence of the composite tag is shown in numbers No. 1 to No. 48. The application method of the label includes: extracting the total DNA of each sample; for each sample, selecting any pair of sequences from 48 pairs of sequences that have not been selected as primers to amplify the total DNA of the corresponding sample; The amplified product of each sample is detected by electrophoresis, and the amplified product is purified and recovered; the purified and recovered product of each sample is accurately quantified by an ultraviolet spectrophotometer; all samples are mixed in equal amounts to establish a sequencing library; Sequencing library for high-throughput sequencing. The invention improves the sample throughput of ultra-high-throughput sequencing, effectively reduces the cost of ultra-high-throughput sequencing, effectively enriches the fungal population in the entire microbial population in the sample, and provides a high-throughput analysis method for the research of fungal diversity in the proven environment .

Description

Composite tags and their applications for high-throughput sequencing of environmental fungal biodiversity

technical field

The invention relates to the technical field of gene sequencing, in particular to a composite tag for high-throughput sequencing of environmental fungal biodiversity, which is used for ultra-high-throughput gene sequencing.

Background technique

Microbial community diversity is one of the focuses of microbial ecology and environmental studies. At present, the use of metagenomics to study microbial diversity has attracted more and more attention from scientists. However, for the same sample, the number of bacteria in the results of metagenomic research often accounts for the majority, making fungi and other microorganisms far less than bacteria. This is very unfavorable for research in some fields, such as the study of microbial populations in various stages of the winemaking process. The role of fungi is much higher than that of bacteria, and has a greater impact on the quality of wine. Therefore, using the unique internal transcribed spacer sequence of fungi as the target sequence, it has become a very effective method to enrich fungal DNA from complex samples by PCR amplification.

Handelman (HandelsmanJ, RondonMR, BradySF.molecularbiologicalaccesstothechemistryofunkownsoilmicrobes:Anewfrontierfornaturalproducts[J].1998,5(10):R245-R249.) et al. (1998) first proposed the concept of micro-genome, which is defined as "thegenomesthetotalmicrobiotafoundinnature" The sum of the genetic material of an organism. It contains the sum of the genomes of both culturable and nonculturable microorganisms. The macro-internal spacer sequence referred to in the present invention refers to the sum of all fungal internal spacer sequences in the environment, which provides a basis for research purposes for microbial diversity, population structure, evolutionary relationship, functional activity, mutual cooperation relationship and relationship with the environment. A new train of thought.

Ribosomal RNA (rRNA) is currently the most widely used molecular marker in microbial molecular ecology because of its highly conserved function and different mutation rates at different positions in the sequence. The genes encoding ribosomal RNA in the eukaryotic genome include 28SrDNA, 5SrDNA, 18SrDNA and 5.8SrDNA. They are connected head to tail on the chromosome, arranged in series, and separated by spacers (Kuang Zhizhou, Xu Yang. Ribosomal rDNAITS sequence Application in mycological research [J]. Chemistry of Life, 2004 (02): 120-122.). Among them, the 18S, 5.8S and 28S rDNA genes constitute a transcription unit, which are highly conserved and suitable for systematic analysis among higher-level biological groups. The spacer in between is the Internal Transcribed Spacer (ITS), including 18S and The internal transcribed spacer 1 (ITS1) between 5.8S and the internal transcribed spacer 2 (ITS2) between 5.8S and 28S, because ITS does not join mature ribosomes, the selection pressure is relatively small, and the evolution rate Faster, showing extremely extensive sequence polymorphism in most eukaryotes. At the same time, the length of the ITS sequence is moderate. The length of the ITS sequence in various eukaryotic organisms from human to yeast ranges from 1000bp to less than 300bp. People can obtain enough information from the sequence that is not too long, and it can be widely used in the genus. Systematic studies of interspecies or intraspecies groups (IwenPC, HinrichsSH, RuppME. Utilization of the internal transcribed spacer regions asmolecular targets to detect and identify human fungal pathogens [J]. MedMycol, 2002, 40 (1): 87-109).

Contents of the invention

The invention provides a composite tag for high-throughput sequencing of environmental fungal biodiversity and its application, which improves the sample throughput of ultra-high-throughput sequencing, effectively reduces the cost of ultra-high-throughput sequencing, and effectively enriches the entire microorganism in the sample The fungal population in the population provides a high-throughput analysis method for the study of fungal diversity in the proven environment.

A composite tag for high-throughput sequencing of environmental fungal biodiversity, the sequence of which is shown in the following sequence numbers NO.1-NO.48.

Each sequence of the composite tag of the present invention is composed of a unique tag consisting of 8-10 bases on the left and a tag consisting of 21 sequences derived from the fungal internal transcriptional spacer 1 on the right.

Table 1

The present invention also provides a fungal high-throughput gene sequencing method, comprising the following steps:

(1) Extract the total DNA of each sample;

(2) For each sample, select any pair of sequences from the 48 pairs of sequences shown in claim 1 that have not been selected as primers to amplify the total DNA of the corresponding sample;

(3) Electrophoresis detection is performed on the amplified product of each sample, and the amplified product is purified and recovered;

(4) Accurate quantification of the product after the purification and recovery of each sample by an ultraviolet spectrophotometer;

(5) Create a sequencing library after mixing all samples in equal amounts;

(6) Perform high-throughput sequencing on the constructed sequencing library.

The fungal macro internal transcriptional spacer sequence of the present invention refers to the sum of all internal transcriptional spacers 1 falling between fungi 18S and 5.8S, and the composite label is as shown in Table 1; the fungal macro internal transcriptional spacer in Table 1 is used The sequence compound tag can achieve the simultaneous detection of the fungal macro-internal spacer sequence in 1 to 48 samples in one ultra-high-throughput sequencing.

Utilizing the current ultra-high-throughput sequencing itself to provide 96 kinds of sequencing tags (8-10 bases), 96 samples can be made, but before ultra-high-throughput sequencing, 96 sequencing libraries are built for each of the 96 samples, and then wait for Quantities are mixed into one and sequenced at the same time, while the present invention can sequence 48 samples at the same time only by building one sequencing library. If the 96 tags included in the sequencing are used in combination with the 48 composite tags of the present invention, ultra-high-throughput sequencing can be performed on the internally transcribed spacer sequences of 96*48 samples at the same time.

In step (1), the total DNA of each sample is extracted using a general extraction method.

In step (2), for each sample, select any pair of sequences from the 48 pairs shown in Table 1 and the sequences that have not been selected as primers. The selection principle is specifically: determine the number of each sample, and for each number Sample, select sequence 1 and sequence 2 corresponding to a sequence number from Table 1 (sequence 1 and sequence 2 of NO.1 are a pair, sequence 1 and sequence 2 of NO.2 are a pair, sequence No.3 1 and sequence 2 are a pair and so on), determine a pair of sequences for each sample; each numbered sample can choose any pair of the 48 pairs in Table 1, but require different numbered samples from Table 1 Do not overlap when selecting, that is, samples with different numbers do not select the same composite label.

The amplification system for amplifying the total DNA of the corresponding sample in step (2) is:

The 50 μL system for PCR amplification contains: 50ng of sample total DNA, 4 μL of Mg ²⁺ , 5 μL of 10×Buffer, 4 μL of 5 nmoldNTP, 1.5 UTaq enzyme, 1.5 μL of each primer at a concentration of 10 pmol, and water to 50 μL.

PCR amplification program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 40°C for 30 s, extension at 72°C for 1 min, 30 cycles, final extension at 72°C for 5 min, and storage at 4°C.

In step (3), the amplification product of each sample is purified and recovered using a general PCR product recovery kit.

The high-throughput sequencing described in the present invention is carried out using a sequencing platform based on the IonPGM second-generation ultra-high-throughput sequencer.

Preferably, the library construction kit used in step (5) is the IonXpress ^™ PlusgDNAFragmentLibraryPreparation kit corresponding to the IonPGM second-generation ultra-high-pass sequencer.

The amplification kit used for amplification during sequencing in step (6) is the IonPGM ^TM TemplateOT2400Kit kit corresponding to the IonPGM second-generation ultra-high-pass sequencer.

The sequencing kit used for sequencing in step (6) is the IonPGMSequencing400Kit kit corresponding to the IonPGM second-generation ultra-high-pass sequencer.

The sample is a fungus-containing sample.

Compared with existing methods, the present invention has the following beneficial effects:

The environment (such as soil) is inhabited by a large number of microorganisms, and the distribution and activities of these microorganisms are closely related to soil nutritional conditions and aboveground vegetation. Wetlands, forests, and oceans are called the three major ecosystems in the world. They are called the "kidneys of the earth" and have rich biodiversity. This method takes microorganisms in a specific environment as the research object, based on the research ideas of metagenomics, using the sequences described in this method, building a library that can perform ultra-high-throughput sequencing on up to 48 samples at the same time, and performing metagenomics-based The research on the change law of fungal populations in environmental samples provides a method for similar research on various environmental samples.

Description of drawings

Fig. 1 is a diagram of the electrophoresis detection result of one of the samples of the present invention.

Figure 2 is an overview of the ultra-high-throughput sequencing results of 8 samples.

detailed description

Example 1

The extraction of the total DNA of environmental samples can refer to the method of Zhou (DNA recovery from soil of diverse composition, Appl. ., 1997, 63, 4993-4995). Or extract according to the method of the soil DNA extraction kit. The main steps are as follows:

1. Weigh 5g of the environmental sample into a 50mL centrifuge tube, add 15ml of phosphate buffer, vortex vigorously on the vortex for 5min, then centrifuge at 12500rpm for 10min, discard the supernatant, and grind the precipitate with sterilized quartz sand;

2. Add 13.5ml DNA extraction buffer, 100μl 20mg/mL proteinase K, shake at 37°C, 250 rpm for 30 minutes;

3. Add 700 microliters of 20% SDS, bathe in 65°C water for 2 hours, shake upside down several times at intervals of 15 minutes;

4. Centrifuge at room temperature for 15 minutes at 6000 rpm, collect the middle liquid phase layer; add 5 ml of DNA extraction solution, 300 microliters of 20% SDS to the precipitate, 65 ° C water bath for 30 min, centrifuge at 6000 rpm, collect the middle liquid phase layer, and combine ;repeat twice;

5. Add an equal volume of chloroform:isoamyl alcohol (24:1) to the collected liquid layer for extraction once, centrifuge at 8000 rpm for 10 min, and collect the upper liquid layer;

6. Add 0.1 times volume of sodium acetate and 0.6 times volume of isopropanol to the collected liquid phase layer in step 5, and place at room temperature for 1-2 hours.

7. Centrifuge overnight sediment at 12,000 rpm for 15 min, discard the supernatant; add 10 mL of cold ethanol to the precipitate for washing, centrifuge at 12,000 rpm for 5 min, discard the supernatant; centrifuge at 12,000 rpm for 30 sec,

8. Naturally dry the precipitate at room temperature, add 400 microliters of TE to the precipitate after drying to dissolve.

DNA extraction solution composition: 100mMTris, 100mM EDTA, 200mMNaCl, 2% PVPP, 3% CTAB, pH9.0

Example 2

Take 8 samples as an example to implement.

(1) Number the 8 samples from 1 to 8, select a sequence number from the No.1 to No.48 sequences in Table 1 (such as Table 2) and submit it to a related sequence synthesis company for synthesis; Dilute the synthesized sequences with water to 10 pmol; correlate the sequence numbers in Table 1 to the corresponding sample numbers, as in Table 2.

Table 2 Correspondence between samples and composite labels

sample number 1 2 3 4 5 6 7 8 serial number No.1 No.2 No.3 No.4 No.5 No.6 No.7 No.8

(2) PCR amplification: Take eight 200 μL PCR tubes, each corresponding to a sample, and use sequence 1 and sequence 2 in the sequence number corresponding to the above sample number as primers for PCR amplification. The 50 μL system for PCR amplification contains: according to the DNA concentration of 8 samples, absorb a total of 50 ng sample total DNA into the corresponding tube, then add 4 μL Mg ²⁺ , 5 μL 10×Buffer, and 4 μL concentration of 5 nmoldNTP to each tube, 1.5 UTaq enzyme, the concentration is 1.5 μL of each primer of 10 pmol, add water to 50 μL.

PCR amplification program: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 40°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, final extension at 72°C for 5 minutes, and storage at 4°C;

(3) Weigh 2 grams of agarose in an Erlenmeyer flask, add water to 100 mL, heat in microwave until completely dissolved, add an appropriate amount of fluorescent display agent and mix well, pour into an electrophoresis mold to cool. Draw 5 μL of the liquid after the above PCR amplification and mix it with the electrophoresis buffer, add it to the sample hole of the agarose gel, and perform constant voltage electrophoresis at a voltage of 5V per cm. After the electrophoresis, put the agarose gel into the ultraviolet Observe the electrophoresis results on the observer, and the successful PCR reaction can be detected by electrophoresis to obtain amplification products with a molecular weight distribution between 200bp and 500bp (as shown in Figure 1).

Example 3

1. Use a general-purpose PCR product recovery kit to purify the PCR amplification product. The steps are as follows:

(1) Add 2 times the volume of BindingBuffer to 1 times the volume of the PCR reaction, turn over and mix well.

(2) Add the above mixture into the DNA purification column, if the volume of the solution is >700μl, transfer the solution in several times; place at room temperature for 1-2min or longer.

(3) Centrifuge at 13000g for 1 minute. After the centrifugation, transfer the solution in the collection tube to the DNA purification column again, centrifuge, and discard the waste liquid.

(4) Add 650 μL WashBuffer to the DNA purification column, centrifuge at 13,000 g for 30 seconds, discard the waste liquid, and put the DNA purification column back into the collection tube. Repeat step 4.

(5) Centrifuge at 13,000g for 3 minutes to remove residual ethanol in the column.

(6) Put the purification column into a new centrifuge tube, add 30-50 μl LutionBuffer or ddH ₂ O preheated at 60°C to the column, and place it at room temperature for 1-2 min or longer.

(7) Centrifuge at 13,000 g for 1 min, and the resulting liquid is high-purity DNA.

2. Accurately quantify the purified PCR amplification product with an ultraviolet spectrophotometer, and adjust the total amount of DNA in the solution with a volume of 70 μL to 100 ng with water;

(8) Use the IonXpress ^™ PlusgDNAFragmentLibraryPreparation kit corresponding to the IonPGM second-generation ultra-high-pass sequencer to build a library;

(9) Use the IonPGM ^TM TemplateOT2400Kit and IonPGMSequencing400Kit kits corresponding to the IonPGM second-generation ultra-high-pass sequencer for sequence amplification and sequencing, 18 chips for sequencing, and input the sequence information of 8 composite tags into the sequencing used by the high-throughput sequencer In the method, the high-throughput sequencer will classify the corresponding sample data according to the input sequence information. The sequencing results obtained according to the above sequencing method (Figure 2) show that the high-throughput sequencer can effectively classify the sequencing data mixed together according to the composite tag sequences corresponding to each sample to obtain the sequencing results of each of the 8 samples. The purpose of composite label application is achieved.

The sequencing method for any other sample size is the same as that in Example 2 and Example 3.

Claims (4)

1. A composite tag for high-throughput sequencing of environmental fungal biodiversity, characterized in that its base sequence is as shown in the following sequence numbers NO.1 to NO.48: 2. A method utilizing the composite label of claim 1 to carry out ultra-high-throughput gene sequencing, characterized in that, comprising the steps of: (1) Extract the total DNA of each sample; (2) For each sample, select any pair of sequences from the 48 pairs of sequences shown in claim 1 that have not been selected as primers to amplify the total DNA of the corresponding sample; (3) Electrophoresis detection is performed on the amplified product of each sample, and the amplified product is purified and recovered; (4) Accurate quantification of the product after the purification and recovery of each sample by an ultraviolet spectrophotometer; (5) Create a sequencing library after mixing all samples in equal amounts; (6) Perform high-throughput sequencing on the constructed sequencing library. 3. The method according to claim 2, wherein the sample is a sample containing fungi. 4. method according to claim 2, is characterized in that, the PCR amplification system that the total DNA of corresponding sample is amplified is: 50ng sample total DNA, 4μL Mg ²⁺ , 5μL 10×Buffer, 4μL concentration of 5nmoldNTP, 1.5UTaq enzyme, concentration of 10pmol primers 1.5μL each, add water to 50μL.