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→‎Sequencing: expanded section on short reads to include illumina and SOLiD, some assembly
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[[File:Environmental shotgun sequencing.png|thumb|250px|Environmental Shotgun Sequencing (ESS). (A) Sampling from habitat; (B) filtering particles, typically by size; (C) DNA extraction and lysis; (D) cloning and library; (E) sequence the clones; (F) sequence assembly.]]
[[File:Environmental shotgun sequencing.png|thumb|250px|Environmental Shotgun
Sequencing (ESS). (A) Sampling from habitat; (B) filtering particles, typically
by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E)
sequencing the clones; (F) sequence assembly into contigs and scaffolds.]]


===Shotgun metagenomics===
===Shotgun metagenomics===
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| doi = 10.1186/1471-2164-7-57
| doi = 10.1186/1471-2164-7-57
| pmid = 16549033
| pmid = 16549033
| pmc = 1483832}}.</ref> This technique for sequencing DNA generates shorter fragments than conventional techniques (~100-200 bp), however this limitation is compensated for by the very large number of sequences generated. In addition, this technique does not require cloning the DNA before sequencing, removing one of the main biases in metagenomics.
| pmc = 1483832}}.</ref> Two other technologies in wide use are the Illumina Genome Analyzer II and the Applied Bio-Systems SOLiD system.<ref name="rodrigue2010">{{cite doi | 10.1371/journal.pone.0011840}}</ref> These techniques for sequencing DNA generate shorter fragments than Sanger sequencing; 454 pyrosequencing typically produces ~100-200 bp reads, Illumina and SOLiD produce 25-75 bp reads.<ref name="schuster2008">{{cite doi | 10.1038/nmeth1156}}</ref> These read lengths are significantly shorter than the typical Sanger sequencing read length of ~750 bp. However, this limitation is compensated for by the much larger number of sequence reads generated. Because most short-read assembly software was not designed for metagenomic applications specialized methods have been developed to utilize mate-read data in metagenomic assembly.<ref name="Charuvaka">{{cite doi | 10.1186/1471-2164-12-S2-S8}}</ref> An additional advantage to short read sequencing is that this technique does not require cloning the DNA before sequencing, removing one of the main biases in environmental sampling.


==Bioinformatics==
==Bioinformatics==

Revision as of 08:02, 28 December 2011

Metagenomics is the study of metagenomes, genetic material recovered directly from environmental samples. The broad field may also be referred to as environmental genomics, ecogenomics or community genomics. While traditional microbiology and microbial genome sequencing rely upon cultivated clonal cultures, early environmental gene sequencing cloned specific genes (often the 16S rRNA gene) to produce a profile of diversity in a natural sample. Such work revealed that the vast majority of microbial biodiversity had been missed by cultivation-based methods.[1] Recent studies use "shotgun" Sanger sequencing or massively parallel pyrosequencing to get largely unbiased samples of all genes from all the members of the sampled communities.[2] Because of its power to reveal the previously hidden diversity of microscopic life, metagenomics offers a powerful lens for viewing the microbial world that has the potential to revolutionize understanding of the entire living world.[3][4]


Etymology

The term "metagenomics" was first used by Jo Handelsman, Jon Clardy, Robert M. Goodman, and others, and first appeared in publication in 1998.[5] The term metagenome referenced the idea that a collection of genes sequenced from the environment could be analyzed in a way analogous to the study of a single genome. Recently, Kevin Chen and Lior Pachter (researchers at the University of California, Berkeley) defined metagenomics as "the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species."[6]

History

Conventional sequencing begins with a culture of identical cells as a source of DNA. However, early metagenomic studies revealed that there are probably large groups of microorganisms in many environments that cannot be cultured and thus cannot be sequenced. These early studies focused on 16S ribosomal RNA sequences which are relatively short, often conserved within a species, and generally different between species. Many 16S rRNA sequences have been found which do not belong to any known cultured species, indicating that there are numerous non-isolated organisms out there.

Early molecular work in the field was conducted by Norman R. Pace and colleagues, who used PCR to explore the diversity of ribosomal RNA sequences.[7] The insights gained from these breakthrough studies led Pace to propose the idea of cloning DNA directly from environmental samples as early as 1985.[8] This led to the first report of isolating and cloning bulk DNA from an environmental sample, published by Pace and colleagues in 1991[9] while Pace was in the Department of Biology at Indiana University. Considerable efforts ensured that these were not PCR false positives and supported the existence of a complex community of unexplored species. Although this methodology was limited to exploring highly conserved, non-protein coding genes, it did support early microbial morphology-based observations that diversity was far more complex than was known by culturing methods.

Soon after that, Healy reported the metagenomic isolation of functional genes from "zoolibraries" constructed from a complex culture of environmental organisms grown in the laboratory on dried grasses in 1995.[10] After leaving the Pace laboratory, Ed DeLong continued in the field and has published work that has largely laid the groundwork for environmental phylogenies based on signature 16S sequences, beginning with his group's construction of libraries from marine samples.[11]

Much of the interest in metagenomics comes from these discoveries that the vast majority of microorganisms had previously gone unnoticed. Traditional microbiological methods relied upon laboratory cultures of organisms. Surveys of ribosomal RNA (rRNA) genes taken directly from the environment revealed that cultivation based methods find less than 1% of the bacteria and archaea species in a sample.[1]

Sequencing

Main article: DNA sequencing

Recovery of DNA sequences longer than a few thousand base pairs from environmental samples was very difficult until recent advances in molecular biological techniques, particularly elated to constructing libraries in bacterial artificial chromosomes (BACs), provided better vectors for molecular cloning.[12]

Environmental Shotgun Sequencing (ESS). (A) Sampling from habitat; (B) filtering particles, typically by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E) sequencing the clones; (F) sequence assembly into contigs and scaffolds.

Shotgun metagenomics

Advances in bioinformatics, refinements of DNA amplification, and proliferation of computational power have greatly aided the analysis of DNA sequences recovered from environmental samples. These advances have enabled the adaptation of shotgun sequencing to metagenomic samples. The approach, used to sequence many cultured microorganisms as well as the human genome, randomly shears DNA, sequences many short sequences, and reconstructs them into a consensus sequence. Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples. This provides information both on which organisms are present and what metabolic processes are possible in the community. This can be helpful in understanding the ecology of a community, particularly if multiple samples are compared to each other.[13] Shotgun metagenomics also is capable of sequencing nearly complete microbial genomes directly from the environment.[14] Because the collection of DNA from an environment is largely uncontrolled, the most abundant organisms in an environmental sample are most highly represented in the resulting sequence data. To achieve the high coverage needed to fully resolve the genomes of underrepresented community members, large samples, often prohibitively so, are needed. On the other hand, the random nature of shotgun sequencing ensures that many of these organisms will be represented by at least some small sequence segments. Due to the limitations of microbial isolation methods, the vast majority of these organisms would go unnoticed using traditional culturing techniques.

In 2002, Mya Breitbart, Forest Rohwer, and colleagues used environmental shotgun sequencing to show that 200 liters of seawater contains over 5000 different viruses.[15] Subsequent studies showed that there are more than a thousand viral species in human stool and possibly a million different viruses per kilogram of marine sediment, including many bacteriophages. Essentially all of the viruses in these studies were new species. In 2004, Gene Tyson, Jill Banfield, and colleagues at the University of California, Berkeley and the Joint Genome Institute sequenced DNA extracted from an acid mine drainage system.[14] This effort resulted in the complete, or nearly complete, genomes for a handful of bacteria and archaea that had previously resisted attempts to culture them. It was now possible to study entire genomes without the biases associated with laboratory cultures.[16]

High-throughput sequencing

In 2006 Robert Edwards, Forest Rohwer, and colleagues at San Diego State University published the first sequences of environmental samples generated with high-throughput sequencing, in this case chip-based pyrosequencing developed by 454 Life Sciences.[17] Two other technologies in wide use are the Illumina Genome Analyzer II and the Applied Bio-Systems SOLiD system.[18] These techniques for sequencing DNA generate shorter fragments than Sanger sequencing; 454 pyrosequencing typically produces ~100-200 bp reads, Illumina and SOLiD produce 25-75 bp reads.[19] These read lengths are significantly shorter than the typical Sanger sequencing read length of ~750 bp. However, this limitation is compensated for by the much larger number of sequence reads generated. Because most short-read assembly software was not designed for metagenomic applications specialized methods have been developed to utilize mate-read data in metagenomic assembly.[20] An additional advantage to short read sequencing is that this technique does not require cloning the DNA before sequencing, removing one of the main biases in environmental sampling.

Bioinformatics

The data generated by metagenomics experiments are both enormous and inherently noisy, containing fragmented data representing as many as 10,000 species.[21] The sequencing of the cow rumen metagenome generated 279 gigabases, or 279 billion base pairs of nucleotide sequence data.[22] Collecting, curating, and extracting useful biological information from datasets of this size represent significant computational challenges for researchers.

Assembly

Main article: Sequence assembly

DNA sequence data from genomic and metagenomic projects are essentially the same, but genomic sequence data offers higher coverage while metagenomic data is usually less redundant.[23] Furthermore, the increased use of second-generation sequencing technologies with short read lengths means that much of future metagenomic data will be error-prone. Taken in combination, these factors make the assembly of metagenomic sequence reads into genomes difficult and unreliable. Misassemblies are caused by the presence of repetitive DNA sequences that make assembly especially difficult because of the difference in the relative abundance of species present in the sample.[24] Missasemblies can also involve the combination of sequences from more than one species into chimeric contigs.

There are several assembly programs, most of which can use information from paired-end tags in order improve the accuracy of assemblies. Some programs, such as Phrap or Celera Assembler, were designed to be used to assemble single genomes but nevertheless produce good results when assembling metagenomic data sets.[21] Other programs, such as Velvet assembler, have been optimized for the shorter reads produced by second-generation sequencing through the use of de Bruijn graphs. The use of reference genomes allows researchers to to improve the assembly of the most abundant microbial species, but this approach is limited by the small subset of microbial phyla for which sequenced genomes are available.[24]

Gene prediction

Main article: Gene prediction

Metagenomic analysis pipelines use two approaches in the annotation of coding regions in the assembled contigs.[24] The first approach is to identify genes based upon homology with genes that are already publicly available in sequence databases, usually by simple BLAST searches. The second, ab initio, uses intrinsic features of the sequence to predict coding regions based upon gene training sets from related organisms. This is the approach taken by programs such as GLIMMER. The main advantage of ab initio prediction is that it enables the detection of coding regions that lack homologs in the sequence databases; however, it is most accurate when there are large regions of contiguous genomic DNA available for comparison.[21]

Species diversity

Gene annotations provide the "what", while measurements of species diversity provide the "who." [25] In order to connect community composition and function in metagenomes, sequences must be binned. Binning is the process of associating a particular sequence with an organism.[24] In similarity based binning, methods such as BLAST are used to rapidly search for phylogenetic markers or otherwise similar sequences in existing public databases; in the case of PhymmBL interpolated Markov models are used to assign reads.[21] In composition based binning, methods use intrinsic features of the sequence, such as oligonucleotide frequencies or codon usage bias.

Databases

The massive amount of exponentially growing sequence data is a daunting challenge that is complicated by the complexity of the metadata associated with metagenomic projects. Metadata includes detailed information about the three-dimensional (including depth, or height) geography and environmental features of the sample, physical data about the sample site, and the methodology of the sampling.[23] This information is necessary both to ensure replicability and to enable downstream analysis. Because of its importance, metadata and collaborative data review and curation require standardized data formats located in specialized databases, such as the Genomes OnLine Database (GOLD).[26]

In 2007, Folker Meyer and Robert Edwards and a team at Argonne National Laboratory and the University of Chicago released the Metagenomics Rapid Annotation using Subsystem Technology server (MG-RAST) a community resource for metagenome data set analysis.[27] As of December 2011 over 9 terabases (9x1012 bases) of DNA have been analyzed, with more than 7,000 public data sets freely available for comparison within MG-RAST. Over 7,000 users now have submitted a total of 38,000 metagenomes to MG-RAST. The Integrated Microbial Genomes/Metagenomes (IMG/M) system also provides a collection of tools for functional analysis of microbial communities based on their metagenome sequence, based upon reference isolate genomes included from the Integrated Microbial Genomes (IMG) system and the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project.[28]

Comparative metagenomics

Comparative analyses between metagenomes can provide additional insight into the function of complex microbial communities and their role in host health.[29] Pairwise or multiple comparisons between metagenomes can be made at the level of sequence composition (comparing GC-content or genome size), taxonomic diversity, or functional complement. Metadata on the environmental context of the metagenomic sample is especially important in comparative analyses, as it provides researchers with the ability to study the effect of habitat upon community structure and function.[21]

Data analysis

Community metabolism

In many bacterial communities, natural or engineered (such as bioreactors), there is significant division of labor in metabolism (Syntrophy), during which the waste products of some organisms are metabolites for others.[30] In one such system, the methanogenic bioreactor, functional stability requires the presence of several syntrophic species (Syntrophobacterales and Synergistia) working together in order to turn raw resources into fully metabolized waste (methane).[31] Using comparative gene studies and expression experiments with microarrays or proteomics researchers can piece together a metabolic network that goes beyond species boundaries. Such studies require detailed knowledge about which versions of which proteins are coded by which species and even by which strains of which species. Therefore, community genomic information is another fundamental tool (with metabolomics and proteomics) in the quest to determine how metabolites are transferred and transformed by a community.[32]

Viruses

Metagenomic sequencing is particularly useful in the study of viral communities. As viruses lack a shared universal phylogenetic marker (as 16S RNA for bacteria and archaea, and 18S RNA for eukarya), the only way to access the genetic diversity of the viral community from an environmental sample is through metagenomics. Viral metagenomes (also called viromes) should thus provide more and more information about viral diversity and evolution.[33]

Case studies

Global Ocean Sampling Expedition

Main article: Global Ocean Sampling Expedition

Beginning in 2003, Craig Venter, leader of the privately-funded parallel of the Human Genome Project, has led the Global Ocean Sampling Expedition (GOS), circumnavigating the globe and collecting metagenomic samples throughout the journey. All of these samples are sequenced using shotgun sequencing, in hopes that new genomes (and therefore new organisms) would be identified. The pilot project, conducted in the Sargasso Sea, found DNA from nearly 2000 different species, including 148 types of bacteria never before seen.[34] Venter has circumnavigated the globe and thoroughly explored the West Coast of the United States, and completed a two-year expedition to explore the Baltic, Mediterranean and Black Seas. Analysis of the metagenomic data collected during this journey revealed two groups of organisms, one composed of taxa adapted to environmental conditions of 'feast or famine', and a second composed of relatively fewer but more abundantly and widely distributed taxa primarily composed of plankton. [35]

Applications

Medicine

Metagenomic sequencing is being used to characterize the microbial communities from 15-18 body sites from at least 250 individuals. This is part of the Human Microbiome initiative with primary goals to determine if there is a core human microbiome, to understand the changes in the human microbiome that can be correlated with human health, and to develop new technological and bioinformatics tools to support these goals.[36]

Environmental remediation

Metagenomics can improve strategies for monitoring the impact of pollutants on ecosystems and for cleaning up contaminated environments. Increased understanding of how microbial communities cope with pollutants is helping assess the potential of contaminated sites to recover from pollution and increase the chances of bioaugmentation or biostimulation trials to succeed.[37]

Biotechtology

Recent progress in mining the rich genetic resource of non-culturable microbes has led to the discovery of new genes, enzymes, and natural products. The impact of metagenomics is witnessed in the development of commodity and fine chemicals, agrochemicals and pharmaceuticals where the benefit of enzyme-catalyzed chiral synthesis is increasingly recognized.[38]

Agriculture

It is well known that the vast majority of microbes have not been cultivated. Functional metagenomics strategies are being used to explore the interactions between plants and microbes through cultivation-independent study of the microbial communities.[39]

See also

References

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  19. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi: 10.1038/nmeth1156, please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi= 10.1038/nmeth1156 instead.
  20. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi: 10.1186/1471-2164-12-S2-S8, please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi= 10.1186/1471-2164-12-S2-S8 instead.
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External links

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