Essay Environmental Shotgun Sequencing: Its Potential and Challenges for Studying the Hidden World of Microbes Jonathan A. Eisen without culturing them [5–7], and taxa may look the same. This vexing the use of high-throughput “shotgun” problem was partially overcome in methods to sequence the genomes of the 1980s through the use of rRNA- cultured species [8]. We are now in the PCR (Table 1). This method allows midst of another such revolution—this microorganisms in a sample to be one driven by the use of genome phylogenetically typed and counted sequencing methods to study microbes based on the sequence of their rRNA directly in their natural habitats, an genes, genes that are present in all approach known as metagenomics, cell-based organisms. In essence, a environmental genomics, or database of rRNA sequences [14,15] ince their discovery in the 1670s community genomics [9]. from known organisms functions by Anton van Leeuwenhoek, In this essay I focus on one like a bird fi eld guide, and fi nding a San incredible amount has been particularly promising area of rRNA-PCR product is akin to seeing a learned about microorganisms and metagenomics—the use of shotgun bird through binoculars. Rather than their importance to human health, genome methods to sequence random counting species, this approach focuses agriculture, industry, ecosystem fragments of DNA from microbes on “phylotypes,” which are defi ned as functioning, global biogeochemical in an environmental sample. The organisms whose rRNA sequences are cycles, and the origin and evolution randomness and breadth of this very similar to each other (a cutoff of of life. Nevertheless, it is what is not environmental shotgun sequencing >97% or >99% identical is frequently known that is most astonishing. For (ESS)—fi rst used only a few years ago used). The ability to use phylotyping example, though there are certainly [10,11] and now being used to assay to determine who was out there in any at least 10 million species of bacteria, every microbial system imaginable microbial sample has revolutionized only a few thousand have been formally from the human gut [12] to waste environmental microbiology [16], described [1]. This contrasts with the water sludge [13]—has the potential to led to many discoveries [e.g.,17], more than 350,000 described species reveal novel and fundamental insights and convinced many people (myself of beetles [2]. This is one of many into the hidden world of microbes and included) to become microbiologists. examples indicative of the general their impact on our world. However, the complexity of analysis required diffi culties encountered in studying Citation: Eisen JA (2007) Environmental shotgun organisms that we cannot readily see to realize this potential poses unique sequencing: Its potential and challenges for studying or collect in large samples for future interdisciplinary challenges, challenges the hidden world of microbes. PLoS Biol 5(3): e82. doi:10.1371/journal.pbio.0050082 analyses. It is thus not surprising that that make the approach both most major advances in microbiology fascinating and frustrating in equal Series Editor: Simon Levin, Princeton University, can be traced to methodological measure. United States of America advances rather than scientifi c Copyright: © 2007 Jonathan A. Eisen. This is an discoveries per se. Who Is Out There? Typing open-access article distributed under the terms and Counting Microbes in of the Creative Commons Attribution License, Examples of these key revolutionary which permits unrestricted use, distribution, and methods (Table 1) include the use of the Environment reproduction in any medium, provided the original microscopes to view microbial cells, One of the most important and author and source are credited. the growth of single types of organisms conceptually straightforward steps Abbreviations: ESS, environmental shotgun in the lab in isolation from other in studying any ecosystem involves sequencing; PCR, polymerase chain reaction; rRNA, ribosomal RNA types (culturing), the comparison cataloging the types of organisms and of ribosomal RNA (rRNA) genes to the numbers of each type. For a long Jonathan A. Eisen is at the University of California construct the fi rst tree of life that time, such typing and counting was Davis Genome Center, with joint appointments in the Section of Evolution and Ecology and included microbes [3], the use of the an almost insurmountable problem in the Department of Medical Microbiology and polymerase chain reaction (PCR) [4] microbiology. This is largely because Immunology, Davis, California, United States of to clone rRNA genes from organisms America. Web site: http://phylogenomics.blogspot. physical appearance does not provide com. E-mail: [email protected] a valid taxonomic picture in microbes. Appearance evolves so rapidly that two This article is part of the Oceanic Metagenomics Essays articulate a specifi c perspective on a topic of collection in PLoS Biology. The full collection is broad interest to scientists. closely related taxa may look wildly available online at http://collections.plos.org/ different and two distantly related plosbiology/gos-2007.php. PLoS Biology | www.plosbiology.org 0001 March 2007 | Volume 5 | Issue 3 | e82 Table 1. Some Major Methods for Studying Individual Microbes Found in the Environment Method Summary Comments Microscopy Microbial phenotypes can be studied by making them more visible. In conjunction The appearance of microbes is not a reliable indicator of with other methods, such as staining, microscopy can also be used to count taxa what type of microbe one is looking at. and make inferences about biological processes. Culturing Single cells of a particular microbial type are grown in isolation from other This is the best way to learn about the biology of a organisms. This can be done in liquid or solid growth media. particular organism. However, many microbes are uncultured (i.e., have never been grown in the lab in isolation from other organisms) and may be unculturable (i.e., may not be able to grow without other organisms). rRNA-PCR The key aspects of this method are the following: (a) all cell-based organisms This method revolutionized microbiology in the 1980s by possess the same rRNA genes (albeit with different underlying sequences); (b) PCR allowing the types and numbers of microbes present in is used to make billions of copies of basically each and every rRNA gene present in a sample to be rapidly characterized. However, there are a sample; this amplifi es the rRNA signal relative to the noise of thousands of other some biases in the process that make it not perfect for all genes present in each organism’s DNA; (c) sequencing and phylogenetic analysis aspects of typing and counting. places rRNA genes on the rRNA tree of life; the position on the tree is used to infer what type of organism (a.k.a. phylotype) the gene came from; and (d) the numbers of each microbe type are estimated from the number of times the same rRNA gene is seen. Shotgun genome The DNA from an organism is isolated and broken into small fragments, and then This has now been applied to over 1,000 microbes, as well sequencing of cultured portions of these fragments are sequenced, usually with the aid of sequencing as some multicellular species, and has provided a much species machines. The fragments are then assembled into larger pieces by looking deeper understanding of the biology and evolution of life. for overlaps in the sequence each possesses. The complete genome can be One limitation is that each genome sequence is usually a determined by fi lling in gaps between the larger pieces. snapshot of one or a few individuals. Metagenomics DNA is directly isolated from an environmental sample and then sequenced. This method allows one to sample the genomes of One approach to doing this is to select particular pieces of interest (e.g., those microbes without culturing them. It can be used both for containing interesting rRNA genes) and sequence them. An alternative is ESS, typing and counting taxa and for making predictions of which is shotgun genome sequencing as described above, but applied to an their biological functions. environmental sample with multiple organisms, rather than to a single cultured organism. doi:10.1371/journal.pbio.0050082.t001 The selective targeting of a single Certainly, many challenges remain the phylotypes and study its properties gene makes rRNA-PCR an effi cient before we can fully realize the potential in the lab. Unfortunately, many, if method for deep community sampling of ESS for the typing and counting of not most, key microbes have not yet [18]. However, this effi ciency comes species, including making automated been cultured [22]. Thus, for many with limitations, most of which are yet accurate phylogenetic trees of every years, the only alternative was to complemented or circumvented by the gene, determining which genes are make predictions about the biology of randomness and breadth of ESS. For most useful for which taxa, combining particular phylotypes based on what example, examination of the random data from different genes even when was known about related organisms. samples of rRNA sequences obtained we do not know if they come from Unfortunately, this too does not work through ESS has already led to the the same organisms, building up well for microbes since very closely discovery of new taxa—taxa that were databases of genes other than rRNA, related organisms frequently have completely missed by PCR because of and making up for the lack of depth of major biological differences. For its inability to sample all taxa equally sampling. If these challenges are met, example, Escherichia coli K12 and E. well (e.g., [19]). In addition, ESS ESS has the potential to rewrite much coli O157:H7 are strains of the same provides the fi rst robust sampling of of what we thought we knew about the species (and considered to be the same genes other than rRNA, and many of phylogenetic diversity of microbial life.