Ulitizing Whole Genome Amplification To Study Metagenomics Of Single Microcystis Colony

Recent years, the crisis of Cyanobacteria blooms in TaihuLake has been more and more serious. Lots of money has been used to purify Taihu Lake, but it works little. The only way to prevent the blooms is to understand the biomechanism of Cyanobacteria. Microcystis colonies play an important role in Cyanobacteria blooms, which Microcystis colonies can resist server environment and absorb nutrition easily. However, the mechanism of formation of colonies is still unknown. The researches of Cyanobacteria mainly focus on groups of Microcystis colonies, however these information of taxonomy and pathway cannot reflect natural conditon of he single colony. As a result, the research on single Microcystis colony is extremely important.Using whole genome amplification make it possible to analysis metagenomics of single Microcystis colony, because the DNA was amplified to meet the requirements of hith-throughput sequencing. Studying the metagenomics of single colony, it is possible to figure out the different of species between different Microcystis species, and find out the key to the formation and different passway of Microcystis colonies. In this article, we focus on Microcystis aeruginosa, Microcystis wesenbergii and Microcystis panniformis, which are most representation and aboundant in Taihu Lake. The main content of the article include the following aspects:1.Utilize microscope to observe the morphology of different single Microcystis colony samples, and pick up different single conlonies. Wash these single colonies to remove the microorganism attatched to the colonies loosely. We use scanning electron microscope to characterize the the surface structure of Microcystis samples in Taihu Lake. We found eukaryote and prokaryote organisms having symbiosis with Microcystis, which helps the bioinformatics analysis.2. We develop a method to isolate and amplify the DNA of single Microcystis conlony, which the product meets the requirement of High-throughput DNA sequencing. Used lysozyme and Proteinase K lysising colony to isolate trace DNA, followed by whole genome amplification to amplify the DNA of single colony, and finally obtained single Microcystis conlony DNA with high quality.3. Construst sequencing library for single Microcystis conlony DNA. To reduce the bias of abundance of different species in different samples, we improve the mass of in-put DNA and reduce the cycle of PCR amplification. Using ultra-sonic to shear DNA, we choose the length of 300-500bp DNA fragments to construct sequencing library under the standard protocols of Illumina. DNA samples were sequenced by HiSeq 4000 platform, and we obtained Pair-End 150bp reads with high quality.4. Analyze High-throughput DNA reads by several bioinformatic tools. Firstly, we aligned sequencing data to protein database in NCBI to obtain the information of different species in different samples, then we construct phylogenetic tree. Removing the data of Microcystis sp, we used principle component analysis tool and clustered the residual data. We found that the same spiece of two Microcystis conlony samples sharing the similar species, and different species of Microcystis conlony samples containing different and special species. Two samples of Microcystis aeruginosa contained Acidobacteria and two samples of Microcystis wesenbergii contained Derxia and Pseudomonadaceae in peculiar,which may related to the development of Microcystis conlonies. What' more, our research showed the advantages in the research of utilizing whole genome amplification to study metagenomics of single Microcystis conlony.