Environmental Shotgun Sequencing Its Potential and Challenges for Studying the Hidden World of Microbes 英文参考文献.docVIP

Essay Environmental Shotgun Sequencing: Its Potential and Challenges for Studying the Hidden World of Microbes Jonathan A. Eisen without culturing them [5–7], and the use of high-throughput “shotgun” methods to sequence the genomes of cultured species [8]. We are now in the midst of another such revolution—this one driven by the use of genome sequencing methods to study microbes directly in their natural habitats, an approach known as metagenomics, environmental genomics, or taxa may look the same. This vexing problem was partially overcome in the 1980s through the use of rRNA- PCR (Table 1). This method allows microorganisms in a sample to be phylogenetically typed and counted based on the sequence of their rRNA genes, genes that are present in all cell-based organisms. In essence, a database of rRNA sequences [14,15] from known organisms functions like a bird ?eld guide, and ?nding a rRNA-PCR product is akin to seeing a bird through binoculars. Rather than counting species, this approach focuses on “phylotypes,” which are de?ned as organisms whose rRNA sequences are very similar to each other (a cutoff of 97% or 99% identical is frequently used). The ability to use phylotyping to determine who was out there in any microbial sample has revolutionized environmental microbiology [16], led to many discoveries [e.g.,17], and convinced many people (myself included) to become microbiologists. S ince their discovery in the 1670s by Anton van Leeuwenhoek, an incredible amount has been learned about microorganisms and their importance to human health, agriculture, industry, ecosystem community genomics [9]. In this essay I focus on one particularly promising area of metagenomics—the use of shotgun genome methods to sequence random fragments of DNA from microbes in an environmental sample. The randomness and breadth of this environmental shotgun sequencing (ESS)—?rst used only a few years ago [10,11] and now being used to assay every microbial system imaginable from the hu