Amplicon sequencing refers to the use of suitable universal primers to amplify the 16S rDNA/18S rDNA/ITS hypervariable regions or functional genes of microorganisms in the environment, and to detect the sequence variation and abundance information of PCR products by high-throughput sequencing technology. Diversity and distribution of microbial communities in the environment to reveal numerous microbial species in environmental samples and their relative abundances and evolutionary relationships.
Scope of application
1. Medical field: the relationship between common diseases and human microorganisms;
2. Animal field: gut, rumen (such as methanogens) and animal health/nutrient digestion research;
3. Agricultural field: rhizosphere microorganisms and Plant interaction, agricultural farming/fertilization treatment and soil microbial community, etc.;
4. Environmental field: haze treatment, sewage treatment, oil degradation, acid mineral water treatment and marine environment, etc.;
5. Special extreme environment: extreme environmental conditions Microbial taxa research.
common problem
1 What is the difference between amplicon sequencing and metagenomic sequencing?
Amplicon sequencing (microbial diversity sequencing) is a high-throughput sequencing of PCR products of specific segments of 16S rDNA/18S rDNA/ITS functional genes, which can determine the composition and abundance of microorganisms in a sample. Metagenome sequencing, by extracting the DNA of all microorganisms in the environment, can not only know the composition and abundance of microorganisms in the sample, but also study the genetic composition and function, metabolism of environmental microorganisms, and discover genes with specific functions, etc.
2 How much data should be measured for each sample, and how many repetitions?
Recommended tags greater than 50,000. If comparative analysis between samples is required, more than 3 intra-group replicates are recommended, and for samples with large individual differences, such as animal feces, more than 10 intra-group replicates are recommended.
3 16S rDNA/18S rDNA/ITS, how to choose the amplified sequence?
Amplicons are mainly divided into 16S, 18S, and ITS sequencing. The 16S sequence is selected for the study of bacterial diversity, the ITS sequence is preferentially selected for fungi, and the 18S is selected for the study of the diversity of eukaryotic microorganisms in the environment.
Classic Case
Simplified and representative bacterial community of maize roots Title: Simplified and representative
bacterial community of maize roots
Publication Journal: PNAS
Publication Date: September 2016
Impact Factor: 9.423
Research Outlines
the Growth and Health of Plant-Associated Microbes tocritical. Results from previous studies have shown that host genotype and abiotic factors influence plant microbial composition. However, the high complexity of these microbial communities presents a challenge to study the composition of microbial communities and the effects of microorganisms on plant hosts. In this work, the authors obtained a greatly simplified synthetic bacterial community consisting of seven strains ( Enterobacter cloacae, Stenotrophomonas maltophilia, Ochrobactrum pituitosum, Herbaspirillum frisingense, Pseudomonas putida, Curtobacterium pusillum , and Chryseobacterium indologenes ) representative of maize Three of the four most dominant phyla found in the root. By using a selective culture-dependent approach to track the abundance of each strain, the authors investigated the effect of different community compositions on maize seedling roots. Only removal of E. cloacae resulted in complete loss of synthetic artificial colonies, which were replaced by C. pusillum . This result suggests that E. cloacae plays a key species role in this model ecosystem. In plants and in vitro, this synthetic bacterial community inhibits the phytopathogenic fungus Fusarium (Fusarium verticillioides ), which showed clear benefits to the host. Therefore, combined with selective culture-dependent quantitative methods, a synthetic seven-species population representing the root microbiota has the potential to serve as a useful system to explore bacterial interspecies interactions and to study the role of microbial communities on the host.
Scheme design
1. Take corn root, rhizosphere and soil samples to detect bacterial communities.
2. Monoclonal maize root bacteria, identify species, simulate the bacterial community composition in the environment, select representative strains, infect aseptically cultured maize roots, and detect the bacterial community composition after infection, and finally select representative strains to construct artificial microbial communities.
3. Eliminate a strain in the artificial microbial community, infect the aseptically cultured corn root, and detect the composition of the colony after infection.
4. Infect the corn root with artificial microbial community and sterile culture with the pathogenic fungus F. verticillioides to observe the fungal infection.
Main results
1. The roots of maize are enriched in some specific genera of Proteobacteria, suggesting that maize roots are enriched in some genera and selectively inhibit some microorganisms (Figure A).
2. Select representative strains to infect maize roots, and detect the distribution of colonies after infection. It is found that the abundance of Enterobacter is obviously dominant. Seven representative strains are selected as artificial microbial communities.
3. Eliminate one strain in the artificial microbial community and infect the aseptically cultured corn root, and found that the artificial microbial community structure can be maintained by removing the other 6 strains. The removal of the E. cloacae strain resulted in the loss of the structure of the synthetic artificial colony, leaving only C. pusillum (Figures B, C).
4. Corn roots with artificial microflora that can resist the fungus Fusarium verticillioidesinfection (Panel D).
