An identification method for aquatic ecological damage assessment based on high-throughput environmental DNA sequencing

Abstract

The application discloses a water ecological damage evaluation identification method based on high-throughput environmental DNA sequencing, and belongs to the technical field of environmental damage investigation and identification, and comprises the following steps: collating a species list of a target area, searching and downloading species sequence information, constructing a local database, and supplementing the local database information through specimen sequencing; collecting benthic animal samples of multiple target area sample points, selecting test samples therefrom, extracting sample DNA, amplifying and performing high-throughput sequencing, performing species comparison according to the sequencing results, presetting multiple read number thresholds, judging whether species in a random mixed species sample and a quantitative proportion mixed species sample are detected, constructing an environmental DNA sequencing and morphological mapping relationship model by using a generalized linear model, optimizing the environmental DNA sequencing and morphological mapping relationship model, calculating the population resource quantity of a specific species in a natural sample, performing water ecological damage indicator species resource quantity evaluation and identification, and improving the water ecological damage identification accuracy and efficiency based on environmental DNA.

Description

Technical Field

[0001] The present application belongs to the technical field of environmental damage investigation and identification, and specifically relates to a method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing. Background Art

[0002] Domestic water quality biological evaluation started late but developed rapidly. In the early 1980s, with the comprehensive development of environmental protection in my country, many scholars began to explore effective methods for water biological monitoring. At present, the development of my country's barcode database is relatively slow. For example, a molecular database for benthic animals has not yet been established, and the established databases do not contain many environmental DNA reference sequences for aquatic organisms in various water bodies.

[0003] At present, water environment monitoring in some areas of my country's river basins only uses surface water indicators as reference. The results can only reflect the instantaneous concentration of water pollutants and cannot reflect the cumulative effect. The traditional water ecological health assessment method based on benthic animals is time-consuming and costly, and cannot effectively support the construction of morphological mapping methods. At the same time, the accuracy and efficiency of water ecological damage identification based on environmental DNA are low, and it is difficult to support and preserve evidence of water ecological damage. Summary of the Invention

[0004] To address the above-mentioned problems and technical deficiencies, the present invention adopts the following technical solution: a method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing, comprising the following steps:

[0005] Step 1: Organize and determine the species list of benthic animals in the target area through literature search; at the same time, conduct benthic animal sampling in the target area and collect benthic animal samples from multiple sampling points;

[0006] Step 2: Based on the species list determined in the previous step, search and download the corresponding species sequence information on the public database website to build a local database;

[0007] If the sequence information of the target benthic species is not retrieved in the public database or the information is incomplete, and samples of the benthic animals are obtained through sampling, the mitochondrial genome sequencing of these samples will be performed separately, and the obtained sequence information will be uploaded and saved in the local database;

[0008] Step 3: According to the benthic animal species composition of the target area, select benthic organisms from the sampled samples to form multiple test samples: random species mixed samples, quantitative species mixed samples and field natural samples;

[0009] Step 4: For all test samples, extract the test sample DNA, use universal primers to amplify the target fragment DNA sequence, perform high-throughput sequencing on the sequence, and select the local database and public database for species comparison of the sequencing results;

[0010] Step 5: Based on the alignment results of the randomly mixed species sample, the read counts of different OTUs of the same species are summed, and multiple read count thresholds are preset. When the summed read count is greater than the threshold, the species is considered to be detected;

[0011] The species mapping rates of the local database and public database of the test sample relative to the actual sample are analyzed according to the OTU relative read number threshold. When the species mapping rate reaches the preset value, the threshold is considered valid.

[0012] Step 6: Based on the comparison results of the quantitatively matched species mixed samples, the read counts of different OTUs of the same species are added together, and the presence of the species is determined based on the read count threshold preset in step 5.

[0013] A generalized linear model was used to construct the correlation between the relative density of each species and the relative number of reads in each target sample, and a relationship model between environmental DNA sequencing and morphological mapping was established;

[0014] Step 7: Select the primer condition with the best model fitting effect from different primer conditions, repeat steps 4 and 6 for the primer multiple times to optimize the relationship model between environmental DNA sequencing and morphology mapping;

[0015] Step 8: Based on the comparison results of natural samples in the wild, combined with the optimized environmental DNA sequencing and morphological mapping relationship model, calculate the population resource quantity information of specific species and conduct an assessment and identification of the resource quantity of water ecological damage indicator species.

[0016] Preferably, the local database includes: basic information of the sample, environmental data and bioinformatics data.

[0017] Furthermore, the basic information of the sample includes: sample number, sampling time, sampling area, sampling point, sampling section, longitude and latitude, sampling depth, habitat type, sampling volume, sample processing method, sample storage location, sampling personnel, recorder and verifier.

[0018] Furthermore, the environmental data are environmental factors at the sampling point, and the environmental factors include physical factors and chemical factors;

[0019] Physical factors include: transparency, pH, temperature, dissolved oxygen, conductivity, turbidity, redox potential, and salinity;

[0020] Chemical factors include: total carbon, total organic carbon, total nitrogen, ammonia nitrogen, nitrate, nitrite, nitrate nitrogen, total phosphorus, and phosphate.

[0021] Furthermore, the bioinformatics data is in the Fastq / Fasta format of the mitochondrial genome sequence of each benthic animal species within the sampling point.

[0022] Preferably, the environmental samples collected from multiple water bodies are collected in a preset amount of benthic animal samples in a target area, and after the species are identified under a stereo microscope and an optical microscope, the complete sequence of the species' mitochondrial genome is obtained by searching a public database or testing the benthic animal samples to construct a local database.

[0023] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0024] This application comprehensively considers the life history characteristics of different types of aquatic organisms, the types of water ecological damage and the areas of occurrence to strengthen the evidentiary capacity and probative force of the water ecological environment damage identification opinion, optimizes the various steps of environmental DNA determination and analysis, and makes breakthroughs in three aspects: sample selection, morphological mapping and physical quantification. First, a local database is constructed and completed, and then the sampling samples are selected to form a variety of test samples. The different read number thresholds of different test samples are compared, and a generalized linear model is used to construct the correlation between the relative density of each species and the relative number of reads in each target sample. A relationship model between environmental DNA sequencing and morphological mapping is established. Finally, for the indicator species of water ecological damage, the quantitative relationship between the population resource quantity and biomass and the detection probability of environmental DNA monitoring is clarified, and a quantitative model is established to infer the population resource quantity and biomass information through the detection probability of environmental DNA monitoring, which strongly supports the construction of morphological mapping methods and the improvement of mapping rates, improves the accuracy and efficiency of water ecological damage identification based on environmental DNA, and better fixes the evidence of water ecological damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In the attached figure:

[0026] Figure 1 A diagram showing the steps of a method according to an embodiment of the present application;

[0027] Figure 2 This is the mitochondrial genome map of Parafossarulus striatulus, a sample from the examples of this application;

[0028] Figure 3 This is the mitochondrial genome map of Alocinma longicornis, a sample from the examples of this application;

[0029] Figure 4This is the mitochondrial genome map of Nephtys oligobranchia, a sample of the present application example;

[0030] Figure 5 This is the mitochondrial genome map of Nereis japonica, a sample of the present application example;

[0031] Figure 6 This is the mitochondrial genome map of Notomastus latericeus, a sample of the present application example;

[0032] Figure 7 This is the mitochondrial genome map of Branchiura sowerbyi, a sample from the examples of this application;

[0033] Figure 8 This is the mitochondrial genome map of Tanypus chinensis, a sample from the examples of this application;

[0034] Figure 9 This is a common mixed species sample test information table of the embodiment of this application;

[0035] Figure 10 This is a test information table of sample of quantitatively proportioned mixtures according to an embodiment of the present application;

[0036] Figure 11 This is the test result table of mixed sample S9# of the embodiment of the present application;

[0037] Figure 12 This is the test result table of mixed sample S10# of the embodiment of the present application;

[0038] Figure 13 This is the quantitative morphological mapping relationship based on the primer mlCOIintF-jgHCO2198 in the embodiment of the present application;

[0039] Figure 14 This is the quantitative morphological mapping relationship based on the primer LCO1490-COIAR in the embodiment of the present application;

[0040] Figure 15 This is the morphological mapping relationship based on five repeated quantifications of primers mlCOIintF-jgHCO2198 in the examples of this application. DETAILED DESCRIPTION

[0041] To make the purposes, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. The components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0042] Embodiments

[0043] As shown in FIG. 1, a water ecological damage assessment identification method based on high-throughput environmental DNA sequencing includes the following steps: Figure 1

[0044] Through literature retrieval, the species list of benthic animals in the target area is sorted and determined; at the same time, benthic animal sampling is carried out in the target area, and benthic animal samples at multiple sampling points are collected;

[0045] According to the determined species list, search and download the corresponding species sequence information on the public database website, and construct a local database;

[0046] If the target benthic species cannot retrieve sequence information or the information is incomplete in the public database, and the benthic animal samples are obtained, the mitochondrial whole genome sequencing is carried out on these samples respectively, and the obtained sequence information is uploaded and saved to the local database;

[0047] The local database includes: basic information of the sample, environmental data, and bioinformatics data.

[0048] The basic information of the sample includes: sample number, sampling time, sampling area, sampling point, sampling section, latitude and longitude, sampling depth, habitat type, sampling volume, sample processing method, sample storage location, sampling personnel, record and verification personnel.

[0049] The environmental data is the environmental factor of the sampling point, and the environmental factor includes physical factor and chemical factor;

[0050] The physical factor includes: transparency, pH, temperature, dissolved oxygen, conductivity, turbidity, oxidation-reduction potential, salinity;

[0051] The chemical factor includes: total carbon, total organic carbon, total nitrogen, ammonia nitrogen, nitrate, nitrite, nitrate nitrogen, total phosphorus, phosphate.

[0052] The bioinformatics data is the mitochondrial genome sequence (Fastq / Fasta format) of each benthic animal species in the sampling point.

[0053] ​Sampling is carried out in the target area, environmental samples from multiple water bodies are collected and uploaded to the local database. After the species are identified under a stereo microscope and an optical microscope, the complete sequence of the species' mitochondrial genome is obtained, specific primers are designed, and a local database is constructed.

[0054] By optimizing the five steps of samples, primers, sequencing, database and result analysis, we aim to achieve an environmental DNA species mapping rate of no less than 85%.

[0055] Test samples were selected from the sampled samples based on species composition;

[0056] Species composition includes three levels: single species, random mixed species, and natural samples.

[0057] Taking Hongze Lake as an example, the dominant species of benthic animals in Hongze Lake were selected, and the main dominant species were Microchironomus tabarui, Chironomus flaviplumus, Propsilocerus akamusi, Tanypus chinensis, Branchiurasowerbyi, Limnodrilus hoffmeisteri, Nephtysoligobranchia, Nereis japonica, Notomastus latericeus, Copper Rust Stone Snail (Nawo), Corbicula fluminea, Parafossarulus striatulus, Alocinma longicornis, Unio douglasiae, and Sinanodontawoodiana. Among them, the species information of the Doba midge, yellow-feathered midge, red-naked midge, Chinese long-legged midge, Hofu water silkworm, copper-rusted stone snail, river clam, round-top pearl oyster and dorsal toothless clam were retrieved in the NCBI database. The information of other species such as Su's tail branch earthworm, oligobranchial toothed sandworm, Japanese spiny sandworm, dorsal earthworm, striped pond snail and long-horned mulch snail is incomplete or missing in the NCBI database, and specimens need to be collected for mitochondrial COI genome sequencing.

[0058] like Figures 2 to 8 As shown, the assembled genome sequence of the sequencing sample is combined with the prediction results of the coding gene to display the sample genome in a circle diagram. These samples are all used to build the data of the local database.

[0059] According to the sampling results of the target area, test samples are selected from the sampling samples, the test samples being respectively common mixed species samples, quantitatively mixed species samples and field natural samples, and a test sample list is as shown in Table 1. Figure 9

[0060] For all test samples, DNA of the test samples is extracted, a target fragment DNA sequence is amplified by using a universal primer, and the sequence is subjected to high-throughput sequencing, and species alignment is performed on the sequencing results respectively in a local database and a public database;

[0061] According to the alignment results of the random mixed species samples, the read numbers of different OTUs of the same species are added, and a plurality of read number thresholds are preset, and when the added read numbers are greater than the threshold, it is considered that the species is detected;

[0062] According to the OTU relative read number threshold, the species mapping rates of the local database and the public database of the test samples relative to the actual samples are analyzed respectively, and when the species mapping rate reaches a preset value, it is considered that the threshold is effective;

[0063] By literature retrieval, a universal primer is selected, DNA sequence information of the target test sample is extracted and amplified, and species alignment is performed on the sequencing results of the environmental DNA of the target test sample respectively in the local database and the public database, different OTU relative read number thresholds are selected by comparison, the morphological mapping rate is improved, and the universal primer corresponding to the target test sample is obtained;

[0064] The process of species alignment is as follows:

[0065] The sample DNA primer sequence information of the target test sample is obtained, the sample DNA primer sequence information of the target test sample is uploaded and saved in the local database, and the local database species mapping rate of the target test sample is obtained according to the DNA primer sequence information of the target test sample in the local database;

[0066] According to the species list of the target test sample, corresponding DNA primer sequence information is searched in the public database, and the public database species mapping rate of the target test sample is obtained according to the DNA primer sequence information of the target test sample in the public database;

[0067] A plurality of OTU relative read number thresholds are preset, and the local database species mapping rate and the public database species mapping rate of the target test sample are compared according to the OTU relative read number threshold, whether the local database species mapping rate meets the mapping rate requirement, whether the public database species mapping rate meets the mapping rate requirement, the condition that the local database species mapping rate meets the mapping rate requirement and the condition that the public database species mapping rate meets the mapping rate requirement are determined.

[0068] As shown in Table 1, the test sample list is as follows:​ Figure 10 As shown, based on the comparison results of the quantitatively matched target species samples, the read numbers of different OTUs of the same species are added together, and the presence of the species is determined based on the above-mentioned preset read number threshold;

[0069] A generalized linear model was used to construct the correlation between the relative density of each species and the relative number of reads in each target sample, and a relationship model between environmental DNA sequencing and morphological mapping was established;

[0070] Quantitatively match the target species and use them as quantitative test samples. Select two universal primers based on the species comparison results, extract and amplify the DNA sequence information of the target test sample, and establish a mapping relationship between environmental DNA sequencing and morphology. Select the primer condition with the best sequencing effect from different primer conditions, further amplify the primer and repeat the sequencing process multiple times to obtain the fitting results again, and analyze and optimize the mapping relationship.

[0071] The universal primers for the target test samples were benthic universal primers mlCOIintF-jgHCO2198. Two known samples of the species specimens were used for environmental DNA sequencing and morphological mapping. The results were as follows: Figure 11 and Figure 12 As shown, the local database contains information on all species, including mitochondrial genomes retrieved from the NCBI database and those tested by this application.

[0072] Mixed sample 9# contains four species of Chironomidae larvae such as Figure 11 The mitochondrial genome sequences of all four Chironomid larvae can be retrieved in the NCBI database, so the comparison results between the local database and the public database (NCBI) are not much different, but from Figure 11 It can still be seen that the local database can achieve a 100% mapping rate when the number of OTU reads is greater than 120, but the comparison results of the public data require that the number of OTU reads be limited to more than 200 to meet the requirement of 80% mapping rate.

[0073] 10# mixed sample is four kinds of mollusks such as Figure 12 The mitochondrial genome sequences of all four species could not be retrieved from the public database (NCBI), but our local database supplemented the missing information. Figure 12 As can be seen, the local database has a better mapping rate than the public database. Furthermore, the local database can achieve a 100% mapping rate when the number of OTU reads is greater than 120, with a low probability of false positives. However, the public database's alignment results cannot meet the 80% mapping rate requirement.

[0074] The results show that building a local database has a significant impact on improving the relationship between environmental DNA sequencing and morphological mapping, especially for some Chinese endemic species, where the information in the public database is more likely to be accurate and requires special attention.

[0075] The process of analyzing and optimizing the morphological mapping relationship is as follows:

[0076] Match the species composition of different target test samples, obtain quantitative morphological mapping relationships under different primer conditions, and obtain the correlation between the relative density of target test samples and the relative read number of the mapped OTUs;

[0077] Select the primer condition with the best sequencing effect from different primer conditions, further amplify the primer and repeat the sequencing process multiple times to obtain the fitting result again;

[0078] The impact of species composition between different target test samples was determined based on the fitting results, and the mapping relationship of high-throughput environmental DNA sequencing was optimized and modified based on the impact results.

[0079] The primer condition with the best model fitting effect is selected from different primer conditions. The above steps of species high-throughput sequencing, species alignment, and construction of relative read number correlation are repeated multiple times for this primer condition to optimize the relationship model between environmental DNA sequencing and morphological mapping.

[0080] Based on the comparison results of natural samples in the wild, combined with the optimized environmental DNA sequencing and morphological mapping relationship model, the population resource quantity information of specific species is calculated, and the resource quantity of water ecological damage indicator species is evaluated and identified.

[0081] The process of establishing the quantitative evaluation model is as follows:

[0082] According to the optimized morphological mapping relationship, an actual community structure information mapping model based on environmental DNA monitoring results was established;

[0083] Obtain sample information of species indicating water ecological damage and input the sample information into the actual community structure information mapping model based on environmental DNA monitoring results;

[0084] Obtain the quantitative relationship between the population resource quantity, biomass and detection probability of environmental DNA monitoring of water ecological damage indicator species;

[0085] Based on the quantitative relationship, a quantitative assessment model is established to infer population resource quantity and biomass information through the detection probability of environmental DNA monitoring.

[0086] Since the mitochondrial genome information of Chironomidae larvae in the public database is relatively sufficient, by matching species combinations of different Chironomidae larvae, Figure 10As shown, the quantitative morphological mapping relationship of different primer conditions was analyzed.

[0087] Depend on Figure 13 and Figure 14 The results show that the relative density of each Chironomid larvae shows a significant positive correlation with the relative read number of the aligned OTU.

[0088] Because the results were based on a single sequencing run, to reduce possible random errors in the sequencing process, we further amplified and repeated the sequencing process 3-6 times to improve the quantitative morphological mapping relationship.

[0089] Since the single sequencing result of primer mlCOIintF-jgHCO2198 was relatively good, this primer was selected for further amplification and the sequencing process was repeated 5 times. The re-fitting results were as follows: Figure 15 shown.

[0090] from Figure 15 It can be seen that the results of the five sequencing runs showed a certain degree of discreteness, which also reflected that there were indeed random errors in the sequencing process. However, from the fitting results, the quantitative relationship of Chironomids chinensis and Chironomids dopaminosa was not significantly improved, but the quantitative results of Chironomids yellow-feathered and Chironomids red naked palp were significantly improved, indicating that considering the influence of random errors can improve the mapping relationship between traditional identification and analysis results and high-throughput environmental DNA sequencing to a certain extent.

[0091] The above-described embodiments merely represent preferred implementations of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present application. It should be noted that a person skilled in the art would be able to make numerous variations, improvements, and substitutions without departing from the spirit of the present application, all of which fall within the scope of protection of the present application.

Claims

1. A method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing, characterized in that: The following steps are involved: Step 1: Organize and determine the species list of benthic animals in the target area through literature search; at the same time, conduct benthic animal sampling in the target area and collect benthic animal samples from multiple sampling points; Step 2: Based on the species list determined in the previous step, search and download the corresponding species sequence information on the public database website to build a local database; If the sequence information of the target benthic species is not retrieved in the public database or the information is incomplete, and samples of the benthic animals are obtained through sampling, the mitochondrial genome sequencing of these samples will be performed separately, and the obtained sequence information will be uploaded and saved in the local database; Step 3: Select benthic organisms from the benthic samples according to the benthic species composition of the target area to form multiple test samples: random species mixed samples, quantitative species mixed samples, and field natural samples; Step 4: For all test samples, extract the test sample DNA, use universal primers to amplify the target fragment DNA sequence, perform high-throughput sequencing on the sequence, and select the local database and public database for species comparison of the sequencing results; Step 5: Based on the comparison results of random species mixed samples, the read counts of different OTUs of the same species are added together, and multiple read count thresholds are preset. When the added read count is greater than the threshold, the species is considered to be detected. The species mapping rates of the local database and public database of the test sample relative to the actual sample are analyzed according to the OTU relative read number threshold. When the species mapping rate reaches the preset value, the threshold is considered valid. Step 6: Based on the comparison results of the quantitatively matched species mixed samples, the read counts of different OTUs of the same species are added together, and the presence of the species is determined based on the multiple read count thresholds preset in step 5; A generalized linear model was used to construct the correlation between the relative density of each species and the relative number of reads in each target sample, and a relationship model between environmental DNA sequencing and morphological mapping was established; Step 7: Select the primer condition with the best model fitting effect from different primer conditions, repeat steps 4 and 6 for the primer multiple times to optimize the relationship model between environmental DNA sequencing and morphology mapping; Step 8: Based on the comparison results of natural samples in the wild, combined with the optimized environmental DNA sequencing and morphological mapping relationship model, calculate the population resource quantity information of specific species and conduct an assessment and identification of the resource quantity of water ecological damage indicator species.

2. The method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing according to claim 1, characterized in that: The local database includes: basic information of samples, environmental data and bioinformatics data.

3. The method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing according to claim 2, characterized in that: The basic information of the sample includes: sample number, sampling time, sampling area, sampling point, sampling section, longitude and latitude, sampling depth, habitat type, sampling volume, sample processing method, sample storage location, sampling personnel, recorder and verifier.

4. The method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing according to claim 2, wherein: The environmental data are environmental factors at the sampling point, which include physical factors and chemical factors; Physical factors include: transparency, pH, temperature, dissolved oxygen, conductivity, turbidity, redox potential, and salinity; Chemical factors include: total carbon, total organic carbon, total nitrogen, ammonia nitrogen, nitrate, nitrite, nitrate nitrogen, total phosphorus, and phosphate.

5. The method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing according to claim 2, characterized in that: The bioinformatics data is the Fastq / Fasta format of the mitochondrial genome sequence of each benthic animal species in the sampling point.

6. The method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing according to claim 1, characterized in that: The method of collecting benthic animal samples at multiple sampling points is to collect a preset amount of benthic animal samples in the target area, identify the species under a stereo microscope and an optical microscope, obtain the complete sequence of the species' mitochondrial genome by searching a public database or testing the benthic animal samples, and construct a local database.

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