CN110894526A - High-throughput sequencing method for benthonic animal COI gene and application thereof - Google Patents
High-throughput sequencing method for benthonic animal COI gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of benthonic animal identification, and particularly relates to a high-throughput sequencing method for a benthonic animal COI gene and application thereof, wherein the high-throughput sequencing method comprises the following steps: s1, carrying out PCR amplification on the COI gene by taking a DNA sample of the benthonic animal mixed sample as a template; s2, carrying out PCR amplification by taking the PCR amplification product in the step S1 as a template, wherein an amplification primer pair comprises a pyrosequencing high-throughput sequencing joint, each sample corresponds to one pyrosequencing high-throughput sequencing joint, and a PCR product is obtained and used as a DNA library; s3, performing quality detection on the obtained DNA library, and then selecting a qualified DNA library to perform emPCR amplification and high-throughput sequencing; the method can obtain the full-length COI gene-658 bp sequence of the large benthonic community with high flux and long read coverage through one-time sequencing, provides higher taxonomic resolution and avoids the problem of false positive caused by overhigh flux.
Description
Technical Field
The invention belongs to the technical field of benthonic animal identification, and particularly relates to a high-throughput sequencing method for a benthonic animal COI gene and application thereof.
Background
The benthic invertebrates generally refer to invertebrate groups living at the bottom of a water body in all or most of life history, and mainly comprise mollusks, aquatic insects, annelids, crustaceans and the like. In water ecology monitoring, benthic invertebrates are essential for evaluating the ecological status of fresh water ecosystems. Although the morphological characteristics play a very important role in species identification, the morphological characteristics are susceptible to limitations of biological sex and specific developmental stages, phenotypic plasticity and genetic variability, and identification errors easily caused by the ubiquitous existence of the cryptospecies and high professional requirements of morphological identification. Scientists expect the total number of biological species to be as much as 1000- & lt1500 & gt over the world, and if the identification is carried out by using the traditional morphological method, the work of all taxonomists all over the world also needs thousands of years to complete the identification. Therefore, new technical methods are urgently needed for species identification, and the DNA barcode technology developed in recent years just meets the requirement.
Cytochrome Oxidase I (COI) generally exists on mitochondria, the gene function is conserved, the gene has a certain evolution rate, the contained genetic evolution information is large, and the Cytochrome oxidase I is widely used for DNA barcode research. The DNA bar code shows the weak side for the mixed sample and the environmental sample, and the second generation sequencing technology solves the problem that the DNA bar code can only identify a single individual. However, the Illumina platform for the second generation sequencing is limited by the read length, mostly based on the-313 bp COI fragment for sequencing, even if the longest read length of 300PE is used, the 658bp COI barcode still has 100bp Gap, the full length of the barcode region cannot be obtained through one-time sequencing, and the existence of the Gap greatly affects the integrity of data, and cannot meet the requirement of species identification. Also, Illumina sequencing is very easy to detect non-target DNA amplicons at very low copy numbers due to the excessively high throughput. The third generation sequencing such as Oxford nanopore and PacBio SMAT has longer reading length, can reduce the splicing cost, but the error rate of single reading length is higher, the sequencing needs to be repeated for error correction, the sequencing cost is increased, the telecommunication software is not rich enough, and the data accumulation is less.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a high-throughput sequencing method for the COI gene of the benthonic animal and application thereof, the sequencing method has high throughput, the data of the COI standard barcode gene-658 bp can be obtained by one-time sequencing, the accuracy is high, and the higher taxonomic resolution is provided.
Therefore, the technical scheme of the invention is as follows:
a high-throughput sequencing method for a benthonic animal COI gene comprises the following steps:
s1, taking a DNA sample of the benthonic animal mixed sample as a template, and carrying out PCR amplification on the COI gene;
s2, carrying out PCR amplification by taking the PCR amplification product in the step S1 as a template, wherein an amplified primer pair comprises a forward primer and a reverse primer, the primer pair comprises a pyrosequencing high-throughput sequencing joint, each sample corresponds to one pyrosequencing high-throughput sequencing joint, and a PCR product is obtained and is used as a DNA library;
s3, performing quality detection on the DNA library obtained in the step S2, and then selecting a qualified DNA library to perform emPCR (emulisionPCR) amplification and high-throughput sequencing.
In the method, in the step S1, the reaction system for PCR amplification is as follows:
10 XBuffer contains 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
forward primer COIF, concentration 5. mu.M, 2. mu.l;
reverse primer COIR, concentration 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
DNA template, 5 ng;
ultrapure water, 35.75. mu.l.
In the method, in the step S1, the reaction procedure of the PCR amplification is as follows: pre-denaturation, keeping at 95 ℃ for 3 min; denaturation, keeping at 95 ℃ for 30s, annealing, keeping at 55 ℃ for 30s, extension, keeping at 72 ℃ for 45s, and circulating for 30-35 times; keeping at 72 deg.C for 7min, and storing at 4 deg.C.
In the method, in the step S2, the reaction system for PCR amplification is as follows:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
454AL + MID + COIF, at a concentration of 5. mu.M, 2. mu.l;
454B + COIR, at a concentration of 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
1ng of DNA template;
ultrapure water, 35.75. mu.l.
In the method, in the step S2, the reaction procedure of the PCR amplification is as follows: pre-denaturation, keeping at 95 ℃ for 3 min; denaturation, keeping at 95 ℃ for 30s, annealing, keeping at 68 ℃ for 30s, extension, keeping at 72 ℃ for 45s, and performing 18 cycles; keeping at 72 deg.C for 7 min; storing at 4 deg.C.
The method, in the step S3, performing quality detection on the library by QC PCR (quantitative PCR), wherein the reaction system of the QC PCR is as follows:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 8. mu.l;
forward primer QC-F, concentration 100. mu.M, 1. mu.l;
reverse primer QC-R, concentration 100. mu.M, 1. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 1. mu.l;
DNA library at a concentration of 1X 108molecules/μl，2μl；
Ultrapure water, 32. mu.l.
The method, in the step S3, detecting the quality of the library by QC PCR, wherein the PCR program of QC PCR is: pre-denaturation, keeping at 94 deg.C for 11 min; denaturation, keeping at 94 deg.C for 1min, annealing, keeping at 60 deg.C for 1min, extension, keeping at 72 deg.C for 1min, and 30 cycles; 72 ℃ for 10 min; storing at 4 deg.C.
In the step S3, exonuclease is added to the reaction system after QC PCR for warm bath, the reaction system before and after the enzyme digestion is electrophoresed, and a DNA library with a sequencing library length of about 750bp is selected as a qualified DNA library.
In the step S3, in the step emPCR amplification, the DNA library is subjected to emPCR lv amplification at a ratio of the number of DNA molecules to the number of DNA capture beads of 3.
The method, in the step S3, further includes a step of performing data analysis on the sequencing result.
A high-throughput sequencing kit for a benthonic animal COI gene comprises a primer pair for amplifying the benthonic animal COI gene, a PCR amplification reaction system, the PCR amplification reaction system and/or a QC PCR reaction system.
The application of the high-throughput sequencing kit for the COI gene of the benthonic animal in obtaining the bar code of the COI gene of the benthonic animal; preferably, the use, including use in water ecology monitoring.
The technical scheme of the invention has the following advantages:
1. the invention provides a high-throughput sequencing method of a benthonic animal COI gene, which comprises the following steps: s1, taking a DNA sample of the benthonic animal mixed sample as a template, and carrying out PCR amplification on the COI gene; s2, carrying out PCR amplification by taking the PCR amplification product in the step S1 as a template, wherein an amplified primer pair comprises a forward primer and a reverse primer, the primer pair comprises a pyrosequencing high-throughput sequencing joint, each sample corresponds to one pyrosequencing high-throughput sequencing joint, and a PCR product is obtained and is used as a DNA library; s3, performing quality detection on the DNA library obtained in the step S2, and then selecting a qualified DNA library to perform emPCR amplification and high-throughput sequencing; the high-throughput sequencing method for the benthonic animal COI gene can obtain a high-throughput reading-length covering large-size benthonic animal community full-length COI gene-658 bp sequence through one-time sequencing, provides higher taxonomic resolution on the basis of guaranteeing high-throughput data, and avoids the problem of false positive caused by overhigh flux.
2. The application of the high-throughput sequencing kit for the COI gene of the benthonic animal provided by the invention in obtaining the bar code of the COI gene of the benthonic animal; the use of (a), including use in water ecology monitoring; the environmental stress factors affecting the large benthonic animals in the Wei river basin are effectively identified as the conductivity and the total nitrogen by the high-throughput sequencing kit and/or the high-throughput sequencing method of the COI genes of the benthonic animals.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a standard graph of accurate quantitative dsDNA based on fluorescence analysis in example 1 of the present invention;
FIG. 2 is a gel map of the detection of COI gene library by Agilent2100 bioanalyzer in example 1 of the present invention;
FIG. 3 is an electrophoresis chromatogram for detecting COI gene library by Agilent2100 bioanalyzer in example 1 of the present invention;
FIG. 4 is a graph plotting the dilution curves of different samples according to example 1 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1
The embodiment provides a high-throughput sequencing method of a benthonic animal COI gene, which comprises the following steps:
first, the sample referred to in this example was prepared as follows: setting representative sampling points according to the characteristics of the geography, the water quality and the like of the water body. Using a Sober net (30 multiplied by 30cm, the aperture of a tuck net is 0.5mm) or a Pederson mud sampler (the opening area is 1/16m2) to collect benthonic animal samples in different habitats, collecting 3 parallel samples at each point, cleaning, roughly picking, putting into a 300mL standard sample bottle, and fixedly storing with 95% alcohol.
(1) The biological mixed sample prepared above was subjected to DNA extraction using a tissue genome DNA extraction kit, and the DNA concentration was measured and diluted in a lump to a final concentration of 5 ng/. mu.l. Taking the DNA with consistent concentration after dilution as a template, wherein a forward primer COIF is shown as SEQ ID NO.1, a reverse primer COIR is shown as SEQ ID NO.2, and carrying out PCR amplification on the COI gene, wherein the reaction system of the PCR amplification is as follows:
10 XBuffer contains 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
forward primer COIF, concentration 5. mu.M, 2. mu.l;
reverse primer COIR, concentration 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
DNA template, 5 ng;
ultrapure water, 35.75 μ l;
the reaction procedure for the PCR amplification is as follows: pre-denaturation, keeping at 95 ℃ for 3 min; denaturation, keeping at 95 ℃ for 30 s; annealing, keeping the temperature at 55 ℃ for 30 s; extending, keeping the temperature at 72 ℃ for 45s, and performing 30-35 cycles; keeping at 72 deg.C for 7min, and storing at 4 deg.C. The amplification product was recovered and quantified on a 1% agarose gel electrophoresis gel.
(2) Taking 1ng of the amplification product recovered in the step (1), performing PCR amplification, wherein an amplified primer pair comprises a forward primer and a reverse primer, the primer pair comprises a pyrosequencing high-throughput sequencing linker, each sample corresponds to a pyrosequencing high-throughput sequencing linker, namely, the forward pyrosequencing high-throughput sequencing linker is added at the 5 'end of a primer COIF, the forward pyrosequencing high-throughput sequencing linker sequence sequentially consists of 454AL sequences and MID sequences in the 5' → 3 'direction, the 454AL sequences are shown in SEQ ID NO.3, the MID sequences consist of 8 arbitrary nucleotides, an MID sequence (shown in a sequence with double underlines in Table 1) is designed for each sample, so that each sample corresponds to one pyrosequencing high-throughput sequencing linker, and a pyrosequencing linker 454B sequence is added at the 5' end of the primer COIR, the 454B sequence is shown as SEQ ID NO.4, the partial primer 454AL + MID (A1-A12) + COIF and the primer 454B + COIR are shown as the following table 1,
TABLE 1 part 454AL + MID (A1-A12) + COIF and primer 454B + COIR
The reaction system of the PCR amplification is as follows:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
454AL + MID + COIF, at a concentration of 5. mu.M, 2. mu.l;
454B + COIR, at a concentration of 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
1ng of DNA template;
ultrapure water, 35.75 μ l;
the reaction procedure for the PCR amplification is as follows: pre-denaturation, keeping at 95 ℃ for 3 min; denaturation, keeping at 95 ℃ for 30 s; annealing, keeping the temperature at 68 ℃ for 30 s; extension, keeping at 72 ℃ for 45s for 18 cycles; keeping at 72 deg.C for 7 min; storing at 4 deg.C. Recovering PCR amplified fragment by glue. The concentration of the recovered PCR amplified fragment was precisely measured using Promega Quant-iT DNA fluorescence detection kit, a standard curve was plotted as shown in FIG. 1, and the DNA molecular copy number was calculated using the following formula, copies/mL ═ 6.022X 1023Copy number/mole) × (concentration ng/mL)/(MW g/mol). Each sample was diluted to 108mu.L of the diluted samples were divided into groups, and 10. mu.L of each of the groups was mixed to prepare a DNA library.
(3) Controlling the quality of the DNA library through QC PCR, wherein a forward primer QC-F is 5'-GGTCAACAAATCATAAAGATATTGG-3', a reverse primer QC-R is 5'-TAAACTTCAGGGTGACCAAAAAATCA-3', and the reaction system of the QC PCR is as follows:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 8. mu.l;
forward primer QC-F, concentration 100. mu.M, 1. mu.l;
reverse primer QC-R, concentration 100. mu.M, 1. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 1. mu.l;
DNA library at a concentration of 1X 108molecules/μl，2μl；
Ultrapure water, 32. mu.l;
the PCR program of the QC PCR is as follows: pre-denaturation, keeping at 94 deg.C for 11 min; denaturing, and keeping at 94 ℃ for 1 min; annealing, and keeping the temperature at 60 ℃ for 1 min; extending, keeping the temperature at 72 ℃ for 1min, and performing 30 cycles; 72 ℃ for 10 min; storing at 4 deg.C.
Mu.l of exonuclease was added to the reaction system after QC PCR and incubated at 37 ℃ for 1h, and 1. mu.l of the system before and after the digestion was run through an Agilent Bioanalyzer 2100(DNA 7500 chip). The sample library without small fragment amplification is a qualified library, and the length of the successfully constructed sequencing library is about 750bp, wherein the sequencing library comprises a sequencing joint and a PCR primer.
Carrying out emPCR amplification and high-throughput sequencing on the qualified DNA library, carrying out emPCR lv amplification on the DNA library according to the ratio of the number of DNA molecules to the number of DNA capture beads being 3, assembling a certain number of DNA capture beads into a sequencing plate, and carrying out high-throughput sequencing. 100 ten thousand valid sequences can be obtained from each sequencing region, and the data amount of each sample is about 100 ten thousand.
Example 2
The sequencing results obtained in example 1 were subjected to data analysis comprising the following steps:
1) and (3) inputting a list file of the MID sequences, operating Split-library in QIIME software to classify the MIDs of different samples, and eliminating degenerate bases, low-quality scores, chimeras and noise sequences shorter than 150bp to obtain pure data.
2) And performing OTU classification on the obtained pure data by using Usearch and CD-HIT software, and performing OTU packing by using a plurality of thresholds of 97%, 98% and the like so as to enhance the reliability of results. OTU representative sequences were matched for species annotation analysis with 80% sequence similarity to sequences in the BOLD database, generating a list of OTUs.
3) Calculating α diversity index such as Shannon index, Simpson index) and Chao index of the sample based on the relative abundance of OTU clustering analysis of the sample was performed using abundance data of the sequence and presence or absence data of OTUs, PCoA analysis of the sample spot for differences of each sample spot was performed using the data coefficients of Jaccard (presence/absence) and Bray-Curtis (relative abundance).
Example 3 Water ecology test application
Environmental stress factors and major pollutants affecting large benthos communities in the watershed were discriminated based on the data obtained in example 2: the water chemistry parameters were normalized via logarithmic transformation. Analyzing the number of classification units of various groups of various types of environmental factors among different groups and the difference of the environmental factors, performing single-factor variance analysis, performing distance-based redundancy analysis (dbRDA), selecting a pre-selection method to gradually delete ineffective environmental variables (P >0.05), screening the environmental stress factors and pollutants influencing the distribution of the large benthonic animal community by a Monte Carlo replacement test method, reflecting the relation between the community distribution and the environmental factors by a sequencing graph, and identifying the main stress factors and pollutants influencing the large benthonic animal community in the drainage basin.
Example 4
The embodiment provides a high-throughput sequencing kit for a benthonic animal COI gene, which comprises a primer pair for amplifying the benthonic animal COI gene, a reaction system for PCR amplification in the step S1, a reaction system for PCR amplification in the step S2 and/or a reaction system for QC PCR.
The primer pair for amplifying the COI gene of the benthonic animal is characterized in that a forward primer COIF is shown as SEQ ID NO.1, and a reverse primer COIR is shown as SEQ ID NO. 2.
The reaction system for PCR amplification in the step S1 is as follows:
10 XBuffer contains 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
forward primer COIF, concentration 5. mu.M, 2. mu.l;
reverse primer COIR, concentration 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
DNA template, 5 ng;
ultrapure water, 35.75. mu.l.
The reaction system for PCR amplification in the step S2 is as follows:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
454AL + MID + COIF, at a concentration of 5. mu.M, 2. mu.l;
454B + COIR, at a concentration of 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
1ng of DNA template;
ultrapure water, 35.75. mu.l.
The QC PCR reaction system is as follows:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 8. mu.l;
forward primer QC-F, concentration 100. mu.M, 1. mu.l;
reverse primer QC-R, concentration 100. mu.M, 1. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 1. mu.l;
DNA library at a concentration of 1X 108molecules/μl，2μl；
Ultrapure water, 32. mu.l.
Example 5
This example provides the application of the method for high-throughput sequencing of the COI gene of the benthonic animal in example 1 or the kit for high-throughput sequencing of the COI gene of the benthonic animal in example 3 in the water ecology monitoring of the Wei river basin, comprising the following steps:
sample collection and water physicochemical parameter measurement: large benthic animal samples are collected aiming at different habitats of the river reach of the Wei river basin, and are fixedly stored by 95 percent ethanol and are brought back to a laboratory to finish the DNA extraction work. Collecting water sample with brown bottle, and treating Total Nitrogen (TN), Total Phosphorus (TP) and ammonia Nitrogen (NH) in water body by national standard method4 +-N), Biological Oxygen Demand (BOD)5) Chemical Oxygen Demand (COD)Mn) And measuring the silicate. The water temperature, pH, conductivity, salinity, dissolved oxygen, suspended matter and total dissolved solids were measured on site using a multifunctional water quality parameter instrument (YSI).
COI standard barcode and morphological identification: large benthic animal samples were collected in duplicate for each spot, one for morphological identification and one for high throughput sequencing based on COI barcodes. Wherein, the morphological identification of the benthonic animals refers to Chinese and local zoology, the high-throughput sequencing method is the same as the embodiment 1, the data analysis method is the same as the embodiment 2, and the aquatic monitoring method is the same as the embodiment 3.
The results of the Weihe river basin aquatic monitoring are shown below, and the representative sequences of OTU were annotated in the BOLD database, and the biological major species include aquatic insects (mayflies, chacopteras, plecopteras, coleopteras), oligotrichomes, mollusks, etc., and 84.7% of the sequences were annotated on the classification order of the genera. Finally, it was identified that conductivity and total nitrogen are significant environmental stress factors affecting the distribution of benthic animal communities in the Wei river basin.
Examples of effects
1. Optimal ratio of DNA molecule number to DNA capture bead
The DNA library of example 1 after passing the test was subjected to an empCR sv (SV EMPCR KIT (LIB-L)) assay to determine the optimal proportional relationship between the number of DNA molecules and the number of DNA capture beads. The number of DNA molecules with the enrichment ratio of 10-20% is the optimal proportion. The effect of the DNA library applied to example 1 in the ratios of the number of DNA molecules to the number of DNA capture beads of 0.5, 1, and 4 is shown in table 2, and it can be seen from the table that the enrichment ratio is 11% when CPB is 1 and 25% when CPB is 4, and in order to ensure a high enrichment ratio and satisfy the optimum ratio, the subsequent emPCR LV (Lib-L) amplification is performed using CPB 3.
TABLE 2 EMPCR sv data sheet
2. The reaction system before exonuclease digestion and after QC PCR in example 1 is detected by an Agilent Bioanalyzer 2100, the gel map is shown in FIG. 2, the electrophoresis chromatogram is shown in FIG. 3, and it can be seen from the gel map that the DNA library prepared in example 1 has no small fragment amplification after quality detection, which shows that the method in example 1 is mature and reliable.
3. The dilution curves of different samples in example 1 are drawn, as shown in fig. 4, the high-throughput sequencing sequence number of the mixed sample of each sampling point reaches the plateau phase, and the sequence number is reasonable, so that the method for high-throughput sequencing of the COI gene of the benthonic animals completely meets the requirements of subsequent research and application under the flux condition.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
SEQUENCE LISTING
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Claims (10)
1. A high-throughput sequencing method for a benthonic animal COI gene is characterized by comprising the following steps:
s1, carrying out PCR amplification on the COI gene by taking a DNA sample of the benthonic animal mixed sample as a template;
s2, carrying out PCR amplification by taking the PCR amplification product in the step S1 as a template, wherein an amplified primer pair comprises a forward primer and a reverse primer, the primer pair comprises a pyrosequencing high-throughput sequencing joint, each sample corresponds to one pyrosequencing high-throughput sequencing joint, and a PCR product is obtained and is used as a DNA library;
and S3, performing quality detection on the DNA library obtained in the step S2, and then selecting a qualified DNA library for empCR amplification and high-throughput sequencing.
2. The method according to claim 1, wherein in the step S1, the reaction system of the PCR amplification is as follows:
10 XBuffer contains 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
forward primer COIF, concentration 5. mu.M, 2. mu.l;
reverse primer COIR, concentration 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
DNA template, 5 ng;
ultrapure water, 35.75. mu.l.
3. The method according to claim 1 or 2, wherein in the step of S1, the reaction procedure of the PCR amplification is as follows: pre-denaturation, keeping at 95 ℃ for 3 min; denaturation, keeping at 95 ℃ for 30s, annealing, keeping at 55 ℃ for 30s, extension, keeping at 72 ℃ for 45s, and circulating for 30-35 times; keeping at 72 deg.C for 7min, and storing at 4 deg.C.
4. The method according to any one of claims 1 to 3, wherein in the step S2, the reaction system of the PCR amplification is as follows:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 4. mu.l;
454AL + MID + COIF, at a concentration of 5. mu.M, 2. mu.l;
454B + COIR, at a concentration of 5. mu.M, 2. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 0.25. mu.l;
1ng of DNA template;
ultrapure water, 35.75. mu.l.
5. The method according to any one of claims 1 to 4, wherein in the step S2, the reaction procedure of the PCR amplification is as follows: pre-denaturation, keeping at 95 ℃ for 3 min; denaturation, keeping at 95 ℃ for 30s, annealing, keeping at 68 ℃ for 30s, extension, keeping at 72 ℃ for 45s, and performing 18 cycles; keeping at 72 deg.C for 7 min; storing at 4 deg.C.
6. A method according to any one of claims 1 to 5, wherein in step S3, the library is quality tested by QC PCR having the reaction system:
10 XBuffer 20mM Mg2+plus，5μl；
dNTP, concentration 2.5mM, 8. mu.l;
forward primer QC-F, concentration 100. mu.M, 1. mu.l;
reverse primer QC-R, concentration 100. mu.M, 1. mu.l;
ex Taq DNA polymerase, at a concentration of 5U/. mu.l, 1. mu.l;
DNA library at a concentration of 1X 108molecules/μl，2μl；
Ultrapure water, 32. mu.l.
7. A method according to any one of claims 1 to 6, wherein in step S3, library quality is detected by QC PCR, which PCR program is: pre-denaturation, keeping at 94 deg.C for 11 min; denaturation, keeping at 94 deg.C for 1min, annealing, keeping at 60 deg.C for 1min, extension, keeping at 72 deg.C for 1min, and 30 cycles; 72 ℃ for 10 min; storing at 4 deg.C.
8. The method according to any one of claims 1 to 7, wherein in the step S3, exonuclease is added to the reaction system after QC PCR for incubation, the reaction system before and after the cleavage is subjected to electrophoresis, and a DNA library having a sequencing library length of about 750bp is selected as a qualified DNA library. Preferably, in the step S3, in the emPCR amplification step, the DNA library is emPCR lv amplified according to a ratio of the number of DNA molecules to the number of DNA capture beads being 3. Preferably, in the step S3, a step of performing data analysis on the sequencing result is further included.
9. A kit for high throughput sequencing of a benthic animal COI gene, comprising a primer pair for amplifying a benthic animal COI gene, a PCR amplification reaction system as claimed in claim 2, a PCR amplification reaction system as claimed in claim 4 and/or a QC PCR reaction system as claimed in claim 6.
10. Use of a high throughput sequencing kit for a benthic animal COI gene according to claim 9 for obtaining a barcode for a benthic animal COI gene; preferably, the use, including use in water ecology monitoring.
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| NL2026403A NL2026403B1 (en) | 2019-09-05 | 2020-09-03 | Next generation sequencing method of zoobenthos cytochrome oxidase subunit i gene and use thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111593099A (en) * | 2020-06-11 | 2020-08-28 | 南京易基诺环保科技有限公司 | Method for monitoring freshwater benthonic animals by utilizing environmental sediment samples |
| CN112359119A (en) * | 2020-11-18 | 2021-02-12 | 中国科学院生态环境研究中心 | Environmental DNA macro-bar code method for large-scale benthonic animal community structure research |
| CN113142034A (en) * | 2021-03-26 | 2021-07-23 | 北京大学 | Method for synchronously identifying floating algae and benthic algae in aquatic ecosystem |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719528A (en) * | 2012-04-27 | 2012-10-10 | 上海派森诺生物科技有限公司 | emPCR method of 454 high-throughput sequencing based on pyrophosphoric acid principle |
| CN104531879A (en) * | 2015-01-06 | 2015-04-22 | 上海海洋大学 | Environment DNA identification method for fish community structure researching |
| CN104630244A (en) * | 2015-01-26 | 2015-05-20 | 中国科学院海洋研究所 | COI gene sequence of species of coral reef benthos and species identification method utilizing COI gene sequence |
| CN106560519A (en) * | 2016-06-11 | 2017-04-12 | 华中农业大学 | Amplimers for mitochondrial COI gene of Bellamya and application thereof |
| CN107746889A (en) * | 2017-07-17 | 2018-03-02 | 国家海洋局北海环境监测中心 | For expanding the primer of benthic polychaeta annelid mitochondria CO I gene fragments |
-
2019
- 2019-09-05 CN CN201910840869.XA patent/CN110894526A/en active Pending
-
2020
- 2020-09-03 NL NL2026403A patent/NL2026403B1/en active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719528A (en) * | 2012-04-27 | 2012-10-10 | 上海派森诺生物科技有限公司 | emPCR method of 454 high-throughput sequencing based on pyrophosphoric acid principle |
| CN104531879A (en) * | 2015-01-06 | 2015-04-22 | 上海海洋大学 | Environment DNA identification method for fish community structure researching |
| CN104630244A (en) * | 2015-01-26 | 2015-05-20 | 中国科学院海洋研究所 | COI gene sequence of species of coral reef benthos and species identification method utilizing COI gene sequence |
| CN106560519A (en) * | 2016-06-11 | 2017-04-12 | 华中农业大学 | Amplimers for mitochondrial COI gene of Bellamya and application thereof |
| CN107746889A (en) * | 2017-07-17 | 2018-03-02 | 国家海洋局北海环境监测中心 | For expanding the primer of benthic polychaeta annelid mitochondria CO I gene fragments |
Non-Patent Citations (6)
| Title |
|---|
| DEINER K, KNAPP RA, BOIANO DM等: "Increased accuracy of species lists developed for alpine lakes using morphology and cytochrome oxidase I for identification of specimens", 《MOLECULAR ECOLOGY RESOURCES》 * |
| HAJIBABAEI M, SHOKRALLA S, ZHOU X等: "Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos", 《PLOS ONE》 * |
| ROCHE: "emPCR Method Manual-Lib-L SV", 《454 SEQUENCING工作手册》 * |
| ROCHE: "GS FLX Titanium emPCR Quick Guide", 《454 SEQUENCING工作手册》 * |
| 李萌，尉婷婷，史博洋等: "环境DNA技术在淡水底栖大型无脊椎动物多样性监测中的应用", 《生物多样性》 * |
| 李飞龙: "DNA barcoding技术在河流生态系统大型底栖动物多样性评估及生物监测中应用研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111593099A (en) * | 2020-06-11 | 2020-08-28 | 南京易基诺环保科技有限公司 | Method for monitoring freshwater benthonic animals by utilizing environmental sediment samples |
| CN112359119A (en) * | 2020-11-18 | 2021-02-12 | 中国科学院生态环境研究中心 | Environmental DNA macro-bar code method for large-scale benthonic animal community structure research |
| CN113142034A (en) * | 2021-03-26 | 2021-07-23 | 北京大学 | Method for synchronously identifying floating algae and benthic algae in aquatic ecosystem |
| CN113142034B (en) * | 2021-03-26 | 2023-02-28 | 北京大学 | Method for synchronously identifying floating algae and benthic algae in aquatic ecosystem |
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| NL2026403B1 (en) | 2022-07-19 |
| NL2026403A (en) | 2021-05-04 |
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