| In recent years,with the deepening of the impact of human activities on the ecological environment,the diversity of species in the environment has gradually become complex and unstable.How to detect the diversity of species in an ecosystem quickly and accurately has become a key process for assessing its stability and establishing ecological concepts and models.The rapid development of modern technology has given rise to the methods of molecular biology,and now,more and more research cannot be done without the help of molecular technology.The technology used environmental DNA combined with high-throughput sequencing to distinguish and identify multiple species is a rapid and efficient method to investigate the abundance of species in a community.In the past decade,people began to use DNA metabarcoding to investigate the biomass of various organisms in various ecological environments gradually.In the research process,the previous experience was summarized and the application of DNA metabarcoding was mainly divided into the following three pairs: organisms in the environment and e DNA,e DNA and PCR product,and the amount of PCR product and high-throughput sequencing results.Thus,the complex methodological research can be divided into three parts which are corresponding to three directions: the interaction between e DNA and the organisms of the environment,the enrichment of e DNA,and the analysis of high-throughput sequencing results.It is the same with quantitative research,and sorting them out clearly is the bright way to study the DNA metabarcoding.The research on the application of DNA metabarcoding to the monitoring of fishery biodiversity,and the solution of some difficult problems such as e DNA enrichment,will be summarized and sorted out into a set of convenient and feasible schemes,which will provide some guidance for practical operation,and be conducive to promoting the application and development of this method in resource survey.At present,the mainstream filtration operations include: choosing different filtration membranes(0.22 and 0.45 ?m respectively)according to the pore size of the filter membrane.According to the filtration methods,there are filtrations using single filtration membranes and using membranes in different pore sizes.In this study,a design experiment was conducted to compare the high-throughput sequencing results of e DNA enriched on the filter membranes of different pore sizes during fractional filtration,so as to analyze the influence of separation and filtration on the detection results.The results showed that the application of fractional filtration was more conducive to the operation.The large tissue particles in the water samples were removed by using large-aperture filter membrane(20 ?m),which could effectively reduce the emergence of "reads with abnormal proportions" in the final results,and did not affect the e DNA enrichment efficiency of small-aperture filter membrane(0.22 ?m).In experimental operation,usually have a certain error,because the instruments and equipment used by artificial operation also has a certain deviation,so inevitably produces the system error,so often the end result is calculated by setting the parallel samples and the mean way to reduce the influence of random error,especially when applied to the quantitative analysis,parallel sample set is very necessary,but the current macro DNA barcode technology application of the determination results of repeatability test is still a lack of direct evidence,so as the research object of this study was to use the same breeding water,respectively with the method of the macro DNA barcode technology do 10 times for detection?The results showed that the test results were close and the repeatability was stable.In the experimental operation,setting 3 parallel samples was enough for analysis and use.To explore the relationship between the resource and the testing results of the bar code technology,a preliminary explore the application of DNA macro method of bar code technology to detect the contents of e DNA in the environment is the key of this method is applied to monitoring the environment resources,although a lot of evidence that e DNA high-throughput sequencing reads several biological and natural environment for relative number correlation,but has been not clear relationship with quantitative results.From environmental biology to laboratory test data,the enrichment efficiency of e DNA is difficult to be evaluated.Meanwhile,primer bias inevitably occurs in the e DNA amplification process,resulting in many uncertainties in the detection results of e DNA macro barcode technology,which restricts the application of e DNA macro barcode technology in the field of biological resource investigation.It was assumed that all e DNA in water was recovered,and there was no primer bias during PCR amplification.In this ideal state,is there a linear relationship between e DNA composition in water and the number of high-throughput sequencing reads?Therefore,in this study,under controlled conditions in the laboratory,two related species of Penaeus vanamei and Penaeus merguiensis were selected,and their DNA samples were mixed in different proportions to simulate e DNA composite samples enriched from natural waters,which not only ensured the recovery of samples,but also reduced the interference of primer bias.Using this as a template,the accuracy of e DNA macro barcoding technique in population relative quantity detection was explored.It was found that when the DNA template concentration ratio of the two species was 1:1,the reads number ratio of the two species interpreted by the high-throughput sequencing results was 13/24(Penaeus merguiensis / Penaeus vanamei).It could be seen that there was still a slight primer bias between the related species of the same genus and the primer migration rate was 1.5%.At the same time,the linear regression analysis between the ratio of reads number of the two species and the ratio of DNA concentration of the two species in the corresponding template obtained by annotation of high-throughput sequencing results obtained by seven test groups indicated that the composition of e DNA in the water showed an obvious linear relationship with the number of high-throughput sequencing reads,that is,y = 0.0716 x + 0.7043(r = 0.9824).It has been preliminary found that the result of HTS is related with the amount of the initial template substrate.It has provide a new way to monitor the biomass in the environment with DNA metabarcoding: when to study the relative amount of species in the specific environment,it can be used that study the quantity of the species in the environment with the analysis method of sequencing results reads which use the ratio between different species. |
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