Water ecological damage assessment and identification method based on high-throughput environmental DNA sequencing

Abstract

The invention discloses a water ecological damage assessment and identification method based on high-throughput environmental DNA sequencing, and belongs to the technical field of environmental damage investigation and identification, and the method comprises the following steps: sorting a species directory of a target area, searching and downloading species sequence information, constructing a local database, and complementing local database information through sample sequencing; the method comprises the following steps: collecting benthonic animal samples of a plurality of target area sample points, selecting a test sample from the benthonic animal samples, extracting DNA of the sample, amplifying, performing high-throughput sequencing, performing species comparison according to a sequencing result, presetting a plurality of reads number thresholds, judging whether species in a random mixture species sample and a quantitatively proportioned species mixed sample are detected or not, and if yes, determining whether the species in the random mixture species sample and the quantitatively proportioned species mixed sample are detected or not. A generalized linear model is adopted to construct an environmental DNA sequencing and morphological mapping relation model, the environmental DNA sequencing and morphological mapping relation model is optimized, the population resource quantity of specific species in a natural sample is calculated, the resource quantity of water ecological damage indication species is evaluated and identified, and the water ecological damage identification accuracy and efficiency based on environmental DNA are improved.

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

[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] In order to solve the above problems and technical defects, the embodiment of the present application 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 the benthic animal samples are obtained through sampling, the mitochondrial whole 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 in the target area, select benthic organisms from the sampled samples to form multiple test samples: random species mixed samples, quantitatively proportioned 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 for the sequencing results;

[0010] Step 5: According to the comparison results of the random mixed species samples, the read numbers of different OTUs of the same species are added, and multiple read number thresholds are preset. When the added read number is greater than the threshold, the species is considered to be detected;

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

[0012] Step 6: According to the comparison results of the quantitatively matched species mixed samples, the read numbers of different OTUs of the same species are added together, and the presence of the species is determined according to the read number 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, selecting the primer condition with the best model fitting effect from different primer conditions, repeating steps 4 and 6 for the primer multiple times, and optimizing the relationship model between environmental DNA sequencing and morphological 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 evaluate and identify 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 of 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 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 beneficial effects of the embodiments of the present application are:

[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, etc. 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 sampled samples are selected to form a variety of test samples. Different reads number thresholds are sampled for different test samples for comparison. 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, and 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 picture:

[0026] Figure 1 A method step diagram of an embodiment of the present application;

[0027] Figure 2 The mitochondrial genome map of Parafossarulus striatulus, a sample of the present application example;

[0028] Figure 3 This is the mitochondrial genome map of the sample Alocinma longicornis of the embodiment of the present 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 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 of the present application example;

[0033] Figure 8 The mitochondrial genome map of Tanypus chinensis, a sample of the present application example;

[0034] Fig. 9 This is a common mixed species sample test information table of the embodiment of the present application;

[0035] Fig.10 This is a test information table of the quantitatively proportioned mixed species samples of the embodiment of the present application;

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

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

[0038] Fig.13 The quantitative morphological mapping relationship based on primer mlCOIintF-jgHCO2198 in the embodiment of the present application;

[0039] Fig.14 The quantitative morphological mapping relationship based on primer LCO1490-COIAR in the embodiment of the present application;

[0040] Fig.15 This is the morphological mapping relationship based on 5 repeated quantifications of primers mlCOIintF-jgHCO2198 in the example of the present application. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Generally, the components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0042] Example

[0043] like Figure 1 As shown, a method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing comprises the following steps:

[0044] By means of literature search, the benthic animal species list of 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 are collected from multiple sampling points;

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

[0046] If the sequence information of the target benthic species is not retrieved in the public database or the information is incomplete, and the benthic animal samples are obtained through sampling, the mitochondrial whole genome sequencing of these samples will be performed separately, and the obtained sequence information will be uploaded and saved in the local database;

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

[0048] 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, recorders and verifiers.

[0049] Environmental data refers to the environmental factors at the sampling point, which include physical factors and chemical factors;

[0050] Physical factors include: transparency, pH, temperature, dissolved oxygen, conductivity, turbidity, redox potential, and salinity;

[0051] Chemical factors include: total carbon, total organic carbon, total nitrogen, ammonia nitrogen, nitrate, nitrite, nitrate nitrogen, total phosphorus, and phosphate.

[0052] The bioinformatics data are the mitochondrial genome sequences (Fastq / Fasta format) of each benthic animal species in the sampling site.

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

[0054] By optimizing the five steps of samples, primers, sequencing, database and result analysis, the goal of achieving an environmental DNA species mapping rate of no less than 85% was achieved;

[0055] The 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, 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 tooth-snout sandworm, Japanese spiny sandworm, dorsal earthworm, striped pond snail and long-horned mullet 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 sampled samples. The test samples are ordinary mixed species samples, quantitative mixed species samples and wild natural samples. The test sample list is as follows: Fig. 9 As shown;

[0060] For all test samples, DNA of the test samples was extracted, and the target fragment DNA sequence was amplified using universal primers. The sequence was sequenced by high-throughput sequencing, and the sequencing results were compared with the local database and the public database for species comparison;

[0061] According to the comparison results of random mixed species samples, the read numbers of different OTUs of the same species are added, and multiple read number thresholds are preset. When the added read number is greater than the threshold, the species is considered to be 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 sample relative to the actual sample are analyzed respectively. When the species mapping rate reaches the preset value, the threshold is considered valid;

[0063] Universal primers were selected through literature retrieval, and the DNA sequence information of the target test samples was extracted and amplified. The environmental DNA sequencing results of the target test samples were compared with local databases and public databases for species comparison. Different OTU relative read number thresholds were selected to improve the morphological mapping rate and obtain universal primers corresponding to the target test samples.

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

[0065] Obtaining sample DNA primer sequence information of the target test sample, uploading and saving the sample DNA primer sequence information of the target test sample to a local database, and obtaining a local database species mapping rate of the target test sample according to the DNA primer sequence information of the target test sample in the local database;

[0066] Retrieving corresponding DNA primer sequence information in a public database according to the species list of the target test sample, and obtaining a public database species mapping rate of the target test sample according to the DNA primer sequence information of the target test sample in the public database;

[0067] Preset multiple OTU relative read number thresholds, and judge and compare the local database species mapping rate and the public database species mapping rate of the target test sample according to the OTU relative read number threshold, to judge whether the local database species mapping rate meets the mapping rate requirements, whether the public database species mapping rate meets the mapping rate requirements, the conditions under which the local database species mapping rate meets the mapping rate requirements, and the conditions under which the public database species mapping rate meets the mapping rate requirements.

[0068] like Fig.10 As shown, according to the comparison results of the target species samples with quantitative matching, the read numbers of different OTUs of the same species are added together, and the presence of the species is determined according to the above-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 the 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, re-obtain the fitting results, 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 are as follows: Fig.11 and Fig.12 As shown, the local database contains information on all species, including the mitochondrial genomes retrieved from the NCBI database and tested by this application.

[0072] Mixed sample 9 contains four types of Chironomid larvae, such as Fig.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 Fig.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 Fig.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. Fig.12 It can be seen that the mapping rate of the local database is better than that of the public database, and the local database can achieve a 100% mapping rate when the number of OTU reads is greater than 120, and the probability of false positives is low. However, the comparison results of the public database 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 the quantitative morphological mapping relationship under different primer conditions, and obtain the correlation between the relative density of the target test samples and the relative reads number of the aligned 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, and the above steps of species high-throughput sequencing, species alignment, and construction of the correlation relationship of relative read numbers are repeated for the primer multiple times 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 water ecological damage indicator species 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 Chironomid larvae in the public database is relatively sufficient, by matching the species combinations of different Chironomid larvae, Fig.10As shown, quantitative morphological mapping relationships of different primer conditions were analyzed.

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

[0088] Because the results are based on a single sequencing, in order to reduce the 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 are as follows: Fig.15 shown.

[0090] from Fig.15 It can be seen that the results of the five sequencings 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 feathers and Chironomids red naked palps 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-mentioned embodiments only express the preferred implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for ordinary technicians in this field, several modifications, improvements and substitutions can be made without departing from the concept of the present application, and these all belong to the protection scope 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 the benthic animal samples are obtained through sampling, the mitochondrial whole 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: According to the benthic animal species composition in the target area, select benthic organisms from the sampled samples to form multiple test samples: random species mixed samples, quantitatively proportioned 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 for the sequencing results; Step 5: According to the comparison results of the random mixed species samples, the read numbers of different OTUs of the same species are added, and multiple read number thresholds are preset. When the added read number is greater than the threshold, the species is considered to be detected; According to the OTU relative read number threshold, the species mapping rates of the local database and the public database of the test sample relative to the actual sample are analyzed respectively. When the species mapping rate reaches the preset value, the threshold is considered valid. Step 6: According to the comparison results of the quantitatively matched species mixed samples, the read numbers of different OTUs of the same species are added together, and the presence of the species is determined according to the read number threshold 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, selecting the primer condition with the best model fitting effect from different primer conditions, repeating steps 4 and 6 for the primer multiple times, and optimizing the relationship model between environmental DNA sequencing and morphological 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 evaluate and identify 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, recorders and verifiers.

4. The method for assessing and identifying water ecological damage based on high-throughput environmental DNA sequencing according to claim 2, characterized in that: The environmental data are environmental factors of the sampling points, 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 environmental samples from multiple water bodies involves collecting a preset amount of benthic animal samples in a target area, identifying the species under a stereo microscope and an optical microscope, and then obtaining the complete sequence of the species' mitochondrial genome by searching a public database or testing the benthic animal samples to construct a local database.

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