Environmental Shotgun Sequencing

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]

S
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 field guide, and finding a
an 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 defined 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)—first 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,
difficulties encountered in studying the complexity of analysis required
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 scientific Copyright: © 2007 Jonathan A. Eisen. This is an
discoveries per se. Who Is Out There? Typing open-access article distributed under the terms
Examples of these key revolutionary and Counting Microbes in of the Creative Commons Attribution License,
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, author and source are credited.
One of the most important and
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,
types (culturing), the comparison ribosomal RNA
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 first 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: jaeisen@ucdavis.edu
a valid taxonomic picture in microbes.
Appearance evolves so rapidly that two This article is part of the Oceanic Metagenomics
Essays articulate a specific 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 amplifies 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 filling 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 efficient 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 efficiency 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 first 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. phylotype), with genomes containing
these genes can be more useful for only about 4,000 genes, yet each
some aspects of typing and counting. What Are They Doing? Top Down possesses hundreds of functionally
Some universal protein coding and Bottom Up Approaches to important genes not seen in the
genes are better than rRNA both for Understanding Functions in other strain [23]. Such differences
distinguishing closely related strains are routine in microbes, and thus one
Communities
(because of third position variation in cannot make any useful inferences
codons) and for estimating numbers A community is, of course, more about what particular phylotypes are
of individuals (because they vary less than a list of types of organisms. doing (e.g., type of metabolism, growth
in copy number between species One approach to understanding properties, role in nutrient cycling, or
than do rRNA genes) [10]. Perhaps the properties and functioning of pathogenicity) based on the activities of
most significantly, ESS is providing a microbial community is to start their relatives.
groundbreaking insights into the with studies of the different types of These difficulties—the inability
diversity of viruses [20,21], which lack organisms and build up from these to culture most microbes and the
rRNA genes and thus were left out of individuals to the community. Ideally, functional disparities between close
the previous revolution. to do this one would culture each of relatives—led to one of the first kinds


Table 2. Methods of Binning
Method Description Comments

Genome assembly Identify regions of overlap between different fragments Getting deep enough sampling for this to work is very expensive
from the same organism to build larger contiguous pieces except for low diversity systems or for very abundant taxa.
(contigs).
Reference genome alignment Identify ESS fragments or contigs that are very similar (a) One of the most effective ways to sort through ESS data, if the
to already assembled sections of the genome of single reference genome is very closely related to an organism in the sample;
microbial types. (b) the reason why more reference genomes are needed; (c) does
not handle regions present in uncultured organisms but not in the
reference.
Phylogenetic analysis Build evolutionary trees of genes encoded by ESS fragments (a) Very powerful, but level of resolution depends on whether
or contigs. Assign fragments or contigs to taxonomic fragments encode useful phylogenetic markers and on how well
groups based on nearest neighbor(s) in trees. sampled the database is for the neighbor analysis; (b) would work
much better if more genomes were available from across the tree of
life.
Word frequency and nucleotide Measure word frequency and composition of each (a) Has the potential to work because organisms sometimes have
composition analysis fragment. Group by clustering algorithms or principal “signatures” of word frequencies that are found throughout the
component analysis. genome and are different between species; (b) very challenging for
small fragments.
Population genetics Build alignments of fragments or contigs with similarity May be most useful as a way of subdividing bins created by other
to each other (but not as much as needed for assembly). methods.
Examine haplotype structure, predicted effective
population size, and synonymous and non synonymous
substitution patterns.

Note that some methods can be applied to ESS fragments or to bins identified by other methods.
doi:10.1371/journal.pbio.0050082.t002

of metagenomic analyses, wherein and species), and these compartments trying to bin? Is it fragments from the
predictions of function were made matter. The key challenge in analyzing same chromosome from a single cell,
from analysis of the sequence of large ESS data is to sort the DNA fragments which would be useful for studying
DNA fragments from representatives (which are usually less than 1,000 base chromosome structure? If so, then
of known phylotypes. This approach pairs long relative to genome sizes of perhaps genome assembly methods
has provided some stunning insights, millions or billions of bases) into bins are the best. What if instead, as in the
such as the discovery of a novel form that correspond to compartments in the sharpshooter example, we are trying to
of phototrophy in the oceans [24]. system being studied. have each bin include every fragment
However, this large insert approach A recent study by myself and that came from a particular species,
has the same limitation as predicting colleagues illustrates the importance knowledge which may be useful for
properties from characterized of compartments when interpreting predicting community metabolic
relatives—a single cell cannot possibly ESS data. When we analyzed ESS data potential? If the level of genetic
represent the biological functions of all from symbionts living inside the gut polymorphism among individual
members of a phylotype. of the glassy-winged sharpshooter (an cells from the same species is high,
ESS provides an alternative, more insect that has a nutrient-limited diet), then genome assembly methods may
global way of assessing biological we were able to bin the data to two not work well (the polymorphisms
functions in microbial communities. As distinct symbionts [26]. We then could will break up assemblies). A better
when using the large insert approach, infer from those data that one of the approach might be to look for species-
functions can be predicted from symbionts synthesizes amino acids for specific “word” frequencies in the
sequences. However, in this case the the host while the other synthesizes DNA, such as ones created by patterns
predicted functions represent a random the needed vitamins and cofactors. in codon usage. The challenge is, how
sampling of those encoded in the Modeling and understanding of this do we tune the methods to find the
genomes of all the organisms present. ecosystem are greatly enhanced by the right target level of resolution? If we
This approach has unquestionably demonstration of this complementary are too stringent, most bins will include
been wildly successful in terms of gene division of labor, in comparison to only a few fragments. But if we are
discovery. For example, analysis of simply knowing that amino acids, too relaxed, we will create artificial
ESS data has revealed novel forms of vitamins, and cofactors are made by constructs that may prove biologically
every type of gene family examined, as “symbionts.” misleading, such as grouping together
well as a great number of completely How does one go about binning sequences from different species. To
novel families (e.g., [25]). However, ESS data? A variety of approaches have make matters more complex, most
there is a major caveat when using been developed, some of which are likely the stringency needed will vary
ESS data to make community-level described in Table 2. In considering for different taxa present in the sample.
inferences. Ecosystems are more than the different binning methods and Another critical issue is the diversity
just a bag of genes—they are made up of their limitations, the first question of the system under study. Generally,
compartments (e.g., cells, chromosomes, one needs to ask is, what are we binning works better when there are


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