Metabarcoding of freshwater communities

hello everyone my name is Roscoe Albrecht and I'm a PhD student at the University perform I work with stream ecosystem assessment and I have developed a method which can use DNA to identify the organisms which live in stream ecosystems in the next few minutes I will show you and demonstrate how this method works freshwater organisms are often used to monitor the health of stream ecosystems unfortunately many of the specimens are difficult to identify based on morphological characters larvae often look very similar even though they belong to different species with different habitat requirements incorrect species identification can lead to incorrect assessment results that means that degraded stream ecosystems might incorrectly be considered as intact vice-versa intact stream ecosystems might be considered as degraded and expensive restoration plans would be employed also that would not be necessary therefore reliable identification of stream organisms is crucial a reliable alternative to morphological identification is DNA barcoding DNA barcoding works like scanning the barcode on a soda-can to identify as a product adult insects can be reliably identified based on morphology in the DNA barcode archived in the database the database can then be used to identify larvae as well as a contain the same DNA barcode as adults unfortunately with DNA barcoding every specimen has to be processed individually making this method too expensive and time-consuming for routine assessments we wanted to use the same DNA based method to identify organisms but we wanted to have a method which could identify hundreds of organisms at once and he is how our method works hundreds of organisms are collected from the stream they are homogenized DNA is extracted and the barcoding fragment copied millions of times millions of DNA barcodes are read by the sequencing machine the barcodes are compared to the database to identify the different species present in the ecosystem the comprehensive taxa list is then used to analyze the health of the stream ecosystem to test the reliability of our laboratory protocols we did run two experiments with ten replications in the first experiment we have tested the influence of specimen size or sequence abundance to test if we also recover sequences of very small stoneflies specimens therefore we used 31 genetically unique specimens of different biomass in the second experiment we tested if our method can detect all of 52 different freshwater specimens at the same time all specimens has similar biomass both experiments succeeded here are the results of the first experiment here you see the number of sequences plotted against specimens wiped the plots show that our method is highly reproducible all ten replicates revealed the same patterns of increasing sequence abundance with specimen biomass all 31 specimens were detected in all replicas even the small specimens small specimens only contributed few sequences to the data set in our second experiment we were able to reliably detect over 80% of the tested species in this plot here you can see in each row the respective species the blue squares indicate that the sample worked in two species was detected if the color is darker more sequences of the organism were detected you can see that the amount of sequences for our species did worry a lot in conclusion we have developed a reliable method for monitoring of freshwater biodiversity in just one single step we were able to detect over 400 specimens and this with higher reliability the morphological identifications we will further improve our protocol and work on integrating it into current ecosystem monitoring programs we are confident that our method will significantly improve the bio monitoring and assessment of aquatic ecosystems in the near future if you want to know more about this project you can visit our website gene stream dot D and now I thank you for watching bye bye

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