CN115161404A - Method for primarily detecting deep-sea movable cold spring by rapidly detecting composition of benthos - Google Patents

Method for primarily detecting deep-sea movable cold spring by rapidly detecting composition of benthos Download PDF

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CN115161404A
CN115161404A CN202210692896.9A CN202210692896A CN115161404A CN 115161404 A CN115161404 A CN 115161404A CN 202210692896 A CN202210692896 A CN 202210692896A CN 115161404 A CN115161404 A CN 115161404A
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何毛贤
姚高友
张华�
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South China Sea Institute of Oceanology of CAS
Southern Marine Science and Engineering Guangdong Laboratory Guangzhou
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Abstract

The invention discloses a method for primarily detecting a deep-sea movable cold spring by quickly detecting the composition of benthic organisms, and belongs to the technical field of oceans. The method comprises the following steps: s1: collecting an environment sample to be detected; s2: filtering an environmental sample and extracting eDNA; s3: performing PCR amplification and high-throughput sequencing on the eDNA; s4: performing OTU clustering according to the determined sequence; s5: comparing NCBInt database, and performing species annotation on OTU sequence; s6: according to the statistical species information of the benthos, preliminarily evaluating whether the locus to be tested is an active cold spring, wherein the evaluation standard of the active cold spring is as follows: the annotation information contains annotation sequences of the deep sea mussels, and the number of reads of the sequences accounts for more than 4% of the total reads of the tested sample. Compared with other existing methods, the method is economical and simple in detection, can detect and identify a large number of deep-sea benthos species at one time at high flux in a target area without frequently using an ROV or an MOV for deep-sea in-situ observation, and preliminarily judges whether the target area is an active cold spring or not according to species composition information.

Description

Method for primarily detecting deep-sea movable cold spring by rapidly detecting composition of benthos
The technical field is as follows:
the invention belongs to the technical field of oceans, and particularly relates to a method for preliminarily detecting a deep-sea movable cold spring by quickly detecting the composition of benthic organisms.
The background art comprises the following steps:
the cold springs are distributed on the edge slope of the active and passive continents below 1000m of the seabed and mainly comprise substances such as water, hydrocarbon mainly comprising methane, hydrogen sulfide and the like. Under a certain temperature and pressure, natural gas hydrate and natural gas stored on the seabed reach an equilibrium state, so that the natural gas diffuses from the deep part of the seabed to the surface of the seabed along a certain channel and overflows upwards to form a movable cold spring. Thus, active cold springs are often accompanied by "combustible ice" (natural hydrates) that is abundant in reserves. The combustible ice is considered as an emerging energy source capable of replacing traditional fossil fuels such as petroleum in the future and has important strategic value. Meanwhile, a large number of cold spring benthos are often found in the active cold spring area, and the deep-sea cold spring benthos contains abundant gene resources, so that the excavation and utilization of low temperature resistance, heavy metal detoxification and other related functional genes can be influenced significantly in the fields of biomedicine, industry, environment, energy and the like.
The main method for detecting the active cold spring at present is to define a potential seabed cold spring area based on early research works such as geological and geophysical surveys, and then to utilize an unmanned deep submersible vehicle (ROV) or a manned deep submersible vehicle (MOV) to examine the potential target sea area one by one, and the method needs to utilize the ROV or the MOV to frequently submerge to the seabed for on-site observation to determine whether the active cold spring is the active cold spring, so that the method has the disadvantages of time consumption, labor consumption, high cost and the like. In addition, the investigation of the diversity of the benthos of the active cold spring mainly comprises the technical means of submarine in-situ shooting, visual identification, indoor one-by-one analysis and identification and the like, and some species which are not easy to be discovered can be omitted by the means; due to the deep water depth (typically over 1000 m), biological individuals are also difficult to acquire, biomass is difficult to count, time consuming and inefficient. Based on the background that the activity cold spring detection and the diversity investigation of the cold spring benthos are time-consuming, high in cost and low in efficiency.
The invention content is as follows:
in order to overcome the problems, the method provided by the invention has the characteristics of high flux and easiness in detection and great reduction of workload, and can be used for preliminarily detecting whether a target area is an active cold spring according to the species composition in a water environment sample by collecting an in-situ bottom layer water sample of a deep sea cold spring area, performing high-flux DNA sequencing on genetic materials in the water sample and identifying the composition of benthic species of the cold spring.
The invention aims to provide a method for primarily detecting deep sea activity cold spring by quickly detecting the composition of benthic organisms, which comprises the following steps:
s1: collecting an environment sample to be detected;
s2: filtering an environmental sample and extracting eDNA;
s3: carrying out PCR amplification and high-throughput sequencing on the eDNA;
s4: performing OTU clustering according to the determined sequence;
s5: comparing NCBI nt database, and performing species annotation on OTU sequence;
s6: according to the statistical species information of the benthos, preliminarily evaluating whether the locus to be detected is an active cold spring, wherein the evaluation standard of the active cold spring is as follows: annotated sequences of deep sea mussel species are present in the sample and the number of reads of the sequences is more than 4% of the total number of reads in the sample tested.
Preferably, the environmental sample in step S1 is a seabed in-situ water environment sample.
Preferably, the environmental sample filtration in step S2 is to filter the processed water sample by using a sterile hydrated cellulose filter membrane with a pore size of 0.45 μm, and store the filtered water sample in liquid nitrogen.
Preferably, the primers for the PCR amplification in step S3 are:
MlCOIintF：5’-GGWACWGGWTGAACWGTWTAYCCYCC-3’；
JghHCO2198：5’-TAIACYTCIGGRTGICCRAARAAYCA-3’。
preferably, the conditions for PCR amplification in step S3 are: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extension for 10min at 72 ℃.
Preferably, the PCR amplification system in step S3 is: 5 XFastpfu Buffer 4. Mu.l, 2.5mM dNTPs 2. Mu.l, forward and reverse primers 0.8. Mu.l each, fastpfu Polymerase 0.4. Mu.l, template DNA 10ng, ddH 2 The amount of O was made up to 20. Mu.l.
Preferably, in the high throughput sequencing in step S3, the raw data-optimized discarding method and parameters are:
(1) Filtering bases with the tail mass value of the read below 20, setting a window of 50bp, if the average mass value in the window is lower than 20, cutting back-end bases from the window, and filtering the read with the mass value below 50bp after quality control;
(2) According to the overlap relation between PE reads, splicing (merge) pairs of reads into a sequence, wherein the minimum overlap length is 10bp;
(3) The maximum mismatch ratio allowed by the overlap region of the spliced sequence is 0.2, and non-compliant sequences are screened out;
(4) Distinguishing samples according to the barcode and the primers at the head end and the tail end of the sequence, and adjusting the sequence direction, wherein the number of mismatch allowed by the barcode is 0, and the maximum number of mismatch of the primers is 2;
(5) Chimeras were removed by de novo and reference binding using Usearch software and gold database.
Preferably, the OTU clustering according to the determined sequence in step S4 is to cluster the high quality sequences at a similarity level of 97% using software Vsearch 2.3.4, resulting in operational classification units (OTUs).
Preferably, the criteria for species annotation of OTU sequences in step S5 are: identity>97% E value<10 -100 。
Compared with the prior art, the invention has the following beneficial effects:
(1) The detection is economical and simple, and ROV or MOV does not need to be frequently used for in-situ observation;
(2) The method can detect and identify a large amount of cold spring benthic species at one time with high flux;
(3) Whether the target area is an active cold spring area or not can be quickly and preliminarily judged.
Drawings
Fig. 1 shows the read count of the living spring living organism deep sea mussel (g. Haimaensisi) at 5 sites H1-H5.
FIG. 2 is ROV in-situ survey and verification, wherein panels A, B and C are respectively active cold spring areas H1, H2 and H3 sites associated with a cold spring biological community mainly comprising a deep sea mussel; panels D and E show the inactive cold spring zones H4 and H5 sites, respectively, and no cold spring benthic organism community was found.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: preliminary detection of active cold springs
1. Firstly, a potential seabed 'cold spring' area is defined based on early research works such as geological and geophysical surveys.
2. Water environment sample collection for potential target area
In the 5 th month in 2021, 5 potential cold spring areas are selected in the southeast jonghai area, and the bottom water of 5 stations to be tested (H1-H5; (16.73 degrees N,110.475 degrees E)) is sampled by a water sampler. Each station has 3 repetitions, each repetition is 1L of water, and 3L of water samples are total. The collected water samples were filtered using sterile cellulose hydration filters (shanghai new asia) with a pore size of 0.45 μm, and after filtration each filter was placed individually in a 25ml centrifuge tube with sterile forceps and stored in liquid nitrogen. The sample environmental DNA (eDNA) was extracted using the OMEGA Water DNAkit (OMEGA, norcross, USA) with reference to the instructions.
3. PCR amplification
PCR amplification is carried out on the extracted eDNA by adopting COI primers. Primer sequences of COI were:
MlCOIintF 5’-GGWACWGGWTGAACWGTWTAYCCYCC-3’；
JghHCO2198，5’-TAIACYTCIGGRTGICCRAARAAYCA-3’。
the PCR reaction system was 4. Mu.l of 5 XFastpfu Buffer, 2. Mu.l of 2.5mM dNTPs, 0.8. Mu.l of each of the forward and reverse primers, 0.4. Mu.l of Fastpfu Polymerase,10ng of eDNA, ddH, and the like 2 The amount of O was made up to 20. Mu.l.
The PCR reaction condition is pre-denaturation at 95 ℃ for 5min; 30s at 95 ℃, 30s at 55 ℃, 45s at 72 ℃ for extension, and 35 cycles; extension at 72 ℃ for 10min. Mixing the PCR products, performing electrophoresis detection with 2% agarose gel, cutting with AxyPrepDNA gel recovery kit (AXYGEN company), recovering PCR products, and eluting with Tris-HCl; and (5) detecting by 2% agarose electrophoresis. Referring to the preliminary quantitative result of electrophoresis, the PCR product is subjected to QuantiFluor TM And carrying out detection and quantification by an ST blue fluorescence quantification system (Promega corporation), and then carrying out Illumina sequencing after mixing according to the corresponding proportion according to the sequencing quantity requirement of each sample.
4. Data optimization process and species annotation
And performing quality control, filtering, splicing and clustering on the original sequencing reads of all samples, and performing NCBI blast comparison on the obtained consistent sequence and a local NT database for taxonomic analysis.
The impurity removal method and parameters for optimizing the high-throughput original sequencing reads data are as follows: (1) Filtering the base with the tail mass value of less than 20 of the read, setting a window of 50bp, if the average mass value in the window is less than 20, cutting off the base at the rear end from the window, and filtering the read with the tail mass value of less than 50bp after quality control; (2) According to the overlap relation between PE reads, splicing (merge) pairs of reads into a sequence, wherein the minimum overlap length is 10bp; (3) The maximum mismatch ratio allowed by overlap region of the splicing sequence is 0.2, and non-conforming sequences are screened out; (4) Distinguishing samples according to the barcode and the primers at the head end and the tail end of the sequence, and adjusting the sequence direction, wherein the permitted mismatch number of the barcode is 0, and the maximum primer mismatch number is 2; (5) Using Usearch software and gold database, remove chimera by de novo and reference combination to obtain high quality sequence.
OTU clustering is to cluster high-quality sequences at a similarity level of 97% by using software Vsearch 2.3.4 to generate Operational Taxonomic Units (OTUs); the species annotation standard is Identity>97% E value<10 -100 。
5. Preliminary evaluation of whether the cold spring is active or not based on biocoenosis composition
The number of deep sea mussels (g. Haimaensis) reads at H1 to H5 sites (in percentage) was 1,586,272 strips (31.72%), 42,355 strips (8.47%), 21,760 (4.35%), 1,884 strips (0.37%) and 1,756 strips (0.35%), respectively (fig. 1). The proportion of deep sea mussel reads in the samples of H1, H2 and H3 is more than 4%, the proportion of deep sea mussel reads in the samples of H4 and H5 sites is far less than 4%, the H1, H2 and H3 sites are preliminarily judged to be active cold spring areas, and the H4 and H5 sites are inactive cold spring areas.
6. ROV in-situ validation survey
ROV in-situ verification investigation is carried out on 5 sites, and whether the target area is an active cold spring or not is judged by taking the standard that obvious plumes are observed on the seabed and a cold spring benthos community is found. ROV survey results show that obvious plumes are observed at H1, H2 and H3 sites, methane signals are obvious, H3 sites are found to have clam (Calyptogena marisinica) communities and a small number of mussel communities, and H1 and H2 sites are found to have a large number of mussel bed biological communities, which belong to typical active cold springs. Although the H4 and H5 sites have methane concentrations, no obvious plume is observed, and no cold spring benthic organism community such as a mussel bed is found. ROV in situ observations further confirmed that H1, H2 and H3 sites were active cold springs (fig. 2).

Claims (9)

1. A method for preliminarily detecting a deep-sea movable cold spring by quickly detecting the composition of benthic organisms is characterized by comprising the following steps:
s1: collecting an environmental sample to be detected;
s2: filtering an environmental sample and extracting eDNA;
s3: carrying out PCR amplification and high-throughput sequencing on the eDNA;
s4: performing OTU clustering according to the determined sequence;
s5: comparing NCBI nt database, and performing species annotation on OTU sequence;
s6: according to the statistical species information of the benthos, preliminarily evaluating whether the locus to be tested is an active cold spring, wherein the evaluation standard of the active cold spring is as follows: annotated sequences from marine mussel species are present in the sample and have a read count of greater than 4% of the total read count of the sample tested.
2. The method of claim 1, wherein the environmental sample of step S1 is a subsea in situ water environmental sample.
3. The method according to claim 1, wherein the environmental sample filtration in step S2 is performed by filtering the treated water sample with a sterile hydrated cellulose filter membrane with a pore size of 0.45 μm and storing in liquid nitrogen.
4. The method of claim 1, wherein the primers for the PCR amplification of step S3 are:
MlCOIintF：5’-GGWACWGGWTGAACWGTWTAYCCYCC-3’；
JghHCO2198：5’-TAIACYTCIGGRTGICCRAARAAYCA-3’。
5. the method of claim 1, wherein the PCR amplification conditions of step S3 are: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extension for 10min at 72 ℃.
6. The method according to claim 1, wherein the PCR amplification system of step S3 is: 5 XFastpfu Buffer 4. Mu.l, 2.5mM dNTPs 2. Mu.l, forward and reverse primers 0.8. Mu.l each, fastpfu Polymerase 0.4. Mu.l, template DNA 10ng, ddH 2 The amount of O was made up to 20. Mu.l.
7. The method of claim 1, wherein the high throughput sequencing of step S3 comprises the following steps, wherein the raw data are optimized by the decontamination method and parameters:
(1) Filtering the base with the tail mass value of less than 20 of the read, setting a window of 50bp, if the average mass value in the window is less than 20, cutting off the base at the rear end from the window, and filtering the read with the tail mass value of less than 50bp after quality control;
(2) According to the overlap relation between PE reads, splicing (merge) pairs of reads into a sequence, wherein the minimum overlap length is 10bp;
(3) The maximum mismatch ratio allowed by the overlap region of the spliced sequence is 0.2, and non-compliant sequences are screened out;
(4) Distinguishing samples according to the barcode and the primers at the head end and the tail end of the sequence, and adjusting the sequence direction, wherein the number of mismatch allowed by the barcode is 0, and the maximum number of mismatch of the primers is 2;
(5) Using Usearch software and gold databases, chimeras were removed by de novo and reference binding.
8. The method according to claim 1, wherein the OTU clustering according to the determined sequences in step S4 is performed by clustering high quality sequences at a similarity level of 97% using software Vsearch 2.3.4, resulting in Operational Taxonomic Units (OTUs).
9. The method according to claim 1, wherein the criteria for species annotation of OTU sequences in step S5 are: identity>97% E value<10 -100 。
CN202210692896.9A 2022-06-17 2022-06-17 Method for primarily detecting deep-sea movable cold spring by rapidly detecting composition of benthos Pending CN115161404A (en)

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