Establishment And Preliminary Application Of Metagenomic Analysis Method For Generating Both Prokaryotic And Eukaryotic Microbial Information

A large number of bacteria,fungi,protozoa and other microorganisms are living in top soil,leaves litters in forest,soils after straw returning to the field,etc.These microorganisms are mainly involving in the degradation of organic matter in the soil and regulating soil nutrients cycles,etc.They often impact the ecological environment of earth,which are also an important part of agriculture and forest ecosystems.Fungi play vital roles in the degradation of forest litter and agricultural straw.However,the existing metagenomic analysis process is basically invented for the human body,which ignores the important eukaryotic microbial information that plays an important role in the cycle of geochemical substances.Currently,no reliable method for the analysis of metagenomic sequencing data of samples such as leaf litter and litter decomposition,etc.In this study,we have successfully established a set of analysis processes that parse all the information of prokaryotic and eukaryotic microorganisms in metagenome and encoding genes through the evaluation and redevelopment of existing bioinformatics analysis piplines.The new analysis system was preliminaryly applied to investigate the influence of acid rain components on the microbial composition of litter decomposition samples.The main results are as follows:1.After comprehensive analysis and comparison,the software Kraken version 2 has the best and more comprehensive results for the analysis of microbial species composition,and most sequences can be marked to species level,which is the most suitable for obtaining information on the species composition of microorganisms from complex metagenomic data.2.Based on Meta Gene Mark and Meta Euk software,a new method for predicting both prokaryotic and eukaryotic coding genes from metagenome has been successfully established.The new method can not only predict the genes of eukaryotes,but also predict the genes of prokaryotes and viruses better and more completely.The method solved the difficulty in predicting both prokaryotic and eukaryotic coding genes from metagenome.3.Prokaryotic genes do not contain introns,while most eukaryotic genes contain introns,it's difficult to quantify their contents in metagenomes.In this study,combination the BLAT software results and self-programming,we established a method to quantify both prokaryotic and eukaryotic encoding genes using Salmon.4.Established a complete set of analysis pipline for analyzing the diversity of prokaryotes and eukaryotes and prediction genes and their functions from metagenomes.5.The results of using LEf Se to determine the differences in key microbial composition are basically consistent with the results of using DESeq2 to obtain different genes and enrichment analysis.6.Acid rain acidity will affect the microbial composition and metabolism in the sample.Low acidity treatment is beneficial to the growth of some eukaryotes,such as Ascomycota.The hyperacidic environment will reduce the abundance of eukaryotes in the sample.