Application of high-throughput DNA sequencing in microbial ecology

Microorganisms play essential roles in natural and artificial ecosystems. However, only a small portion of microorganisms can be cultured and isolated in most ecosystems, which greatly limits our understanding of microbial community composition, ecological function and their potential applications. The novel molecular techniques, especially high-throughput sequencing, have provided advantages for further exploring the diversity, composition and ecological functions of microbial community. High-throughput sequencing is a culture-independent technology capable of deep, rapid and accurate detection of genetic information. With the update of the high-throughput sequencing technology, the sequencing throughput, read length and accuracy have been dramatically improved, and the cost has greatly declined. During the past decade, high-throughput sequencing technology has been rapidly applied in the microbial ecological studies with various types of samples such as soil, water and gut microbial communities. Amplicon and metagenomic sequencing are most widely used strategies for environmental samples. Amplicon sequencing refers to sequencing the PCR products of target gene fragments amplified with specifically designed primers. The 16S rRNA gene of prokaryotes and the 18S rRNA and ITS genes of eukaryotes are most commonly used marker genes to conduct microbial taxonomic analysis. In addition, functional genes such as nirK, nirS and nosZ genes of denitrifying bacteria, amoA gene of ammonia-oxidizing bacteria, and ureC gene of urea hydrolytic bacteria are frequently adopted to study the diversity of functional microorganisms. In metagenomics, sequencing is performed on the genomic DNA directly extracted from environmental samples therefore avoiding the bias from PCR. Theoretically this method can provide a representation of all genomes in the sample and can be used for fully exploring the genetic diversity, functional potentials and metabolic pathways of both cultured and uncultured microorganisms. Metagenomic sequencing technology has been applied in the field of medical diagnosis, human health, biological energy, environmental restoration and agricultural ecology, etc. and has provided us new insights into taxonomic diversity, ecological function, evolutionary succession and interaction in the complex microflora. The application of metagenomic sequencing technology to the field of virology is referred as viral metagenomics. Viruses are not only related to various diseases of crop, animal and human, but also indispensable in the natural ecosystems, and play an important role in regulating host diversity and community succession, mediating gene transfer between microbes, and promoting global biogeochemical cycles. Viral metagenomics has recently gained momentum in application in the field of environmental science to reveal the genetic diversity, explore the novel species of viruses, and investigate their interactions with environmental factors.