metagenomics metagenomics metagenomics is the study of genetic material recovered directly from environmental or clinical samples by a method called sequencing. the broad field may also be referred to as environmental genomics, ecogenomics, community genomics or microbiomics. part of a series ongenetics key components chromosome dna rna genome heredity mutation nucleotide variation outline index history and topics introduction history evolution (molecular) population genetics mendelian inheritance quantitative genetics molecular genetics research geneticist dna sequencing genetic engineering genomics ( template) medical genetics branches of genetics personalized medicine personalized medicine category in metagenomics, the genetic materials (dna, c) are extracted directly from samples taken from the environment (e.g. soil, sea water, human gut, a) after filtering (b), and are sequenced (e) after multiplication by cloning (d) in an approach called shotgun sequencing. these short sequences can then be put together again using assembly methods (f) to deduce the individual genomes or parts of genomes that constitute the original environmental sample. this information can then be used to study the species diversity and functional potential of the microbial community of the environment. while traditional microbiology and microbial genome sequencing and genomics 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. because of its ability 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. as the price of dna sequencing continues to fall, metagenomics now allows microbial ecology to be investigated at a much greater scale and detail than before. recent studies use either "shotgun" or pcr directed sequencing to get largely unbiased samples of all genes from all the members of the sampled communities. etymology the term "metagenomics" was first used by jo handelsman, robert m. goodman, michelle r. rondon, jon clardy, and sean f. brady, and first appeared in publication in 1998. 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. in 2005, kevin chen and lior pachter (researchers at the university of california, berkeley) defined metagenomics as "the application of modern genomics technique without the need for isolation and lab cultivation of individual species". history part of a series ondna barcoding dna barcoding • metabarcoding by taxa microbial fungal pollen algae aquatic macroinvertebrates fish other environmental dna (edna) environmental rna metagenomics viruses metatranscriptomics amplification pcr shotgun sequencing high throughput sequencing extracellular rna chimera healthcare diet assessment consortium for the barcode of life category 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 (rrna) 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. these surveys of ribosomal rna genes taken directly from the environment revealed that cultivation based methods find less than 1% of the bacterial and archaeal species in a sample. much of the interest in metagenomics comes from these discoveries that showed that the vast majority of microorganisms had previously gone unnoticed. in the 1980s 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. the insights gained from these breakthrough studies led pace to propose the idea of cloning dna directly from environmental samples as early as 1985. this led to the first report of isolating and cloning bulk dna from an environmental sample, published by pace and colleagues in 1991 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 in 1995, 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. after leaving the pace laboratory, edward 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. in 2002, mya breitbart, forest rohwer, and colleagues used environmental shotgun sequencing (see below) to show that 200 liters of seawater contains over 5000 different viruses. 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. 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. 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 were 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. venter 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. in 2005 stephan c. schuster at penn state university and colleagues published the first sequences of an environmental sample generated with high-throughput sequencing, in this case massively parallel pyrosequencing developed by 454 life sciences. another early paper in this area appeared in 2006 by robert edwards, forest rohwer, and colleagues at san diego state university. sequencing flow diagram of a typical metagenome project recovery of dna sequences longer than a few thousand base pairs from environmental samples was very difficult until recent advances in molecular biological techniques allowed the construction of libraries in bacterial artificial chromosomes (bacs), which provided better vectors for molecular cloning. shotgun metagenomics advances in bioinformatics, refinements of dna amplification, and the proliferation of computational power have greatly aided the analysis of dna sequences recovered from environmental samples, allowing the adaptation of shotgun sequencing to metagenomic samples (known also as whole metagenome shotgun or wmgs sequencing). the approach, used to sequence many cultured microorganisms and the human genome, randomly shears dna, sequences many short sequences, and reconstructs them into a consensus sequence. shotgun sequencing reveals genes present in environmental samples. historically, clone libraries were used to facilitate this sequencing. however, with advances in high throughput sequencing technologies, the cloning step is no longer necessary and greater yields of sequencing data can be obtained without this labour-intensive bottleneck step. shotgun metagenomics provides information both about which organisms are present and what metabolic processes are possible in the community. 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 under-represented 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, which would otherwise go unnoticed using traditional culturing techniques, will be represented by at least some small sequence segments. high-throughput sequencing an advantage to high throughput sequencing is that this technique does not require cloning the dna before sequencing, removing one of the main biases and bottlenecks in environmental sampling. the first metagenomic studies conducted using high-throughput sequencing used massively parallel 454 pyrosequencing. three other technologies commonly applied to environmental sampling are the ion torrent personal genome machine, the illumina miseq or hiseq and the applied biosystems solid system. these techniques for sequencing dna generate shorter fragments than sanger sequencing; ion torrent pgm system and 454 pyrosequencing typically produces ~400 bp reads, illumina miseq produces 400-700bp reads (depending on whether paired end options are used), and solid produce 25–75 bp reads. historically, these read lengths were significantly shorter than the typical sanger sequencing read length of ~750 bp, however the illumina technology is quickly coming close to this benchmark. however, this limitation is compensated for by the much larger number of sequence reads. in 2009, pyrosequenced metagenomes generate 200–500 megabases, and illumina platforms generate around 20–50 gigabases, but these outputs have increased by orders of magnitude in recent years. an emerging approach combines shotgun sequencing and chromosome conformation capture (hi-c), which measures the proximity of any two dna sequences within the same cell, to guide microbial genome assembly. long read sequencing technologies, including pacbio rsii and pacbio sequel by pacific biosciences, and nanopore minion, gridion, promethion by oxford nanopore technologies, is another choice to get long shotgun sequencing reads that should make ease in assembling process. bioinformatics schematic representation of the main steps necessary for the analysis of whole metagenome shotgun sequencing-derived data. the software related to each step is shown in italics. the data generated by metagenomics experiments are both enormous and inherently noisy, containing fragmented data representing as many as 10,000 species. the sequencing of the cow rumen metagenome generated 279 gigabases, or 279 billion base pairs of nucleotide sequence data, while the human gut microbiome gene catalog identified 3.3 million genes assembled from 567.7 gigabases of sequence data. collecting, curating, and extracting useful biological information from datasets of this size represent significant computational challenges for researchers. sequence pre-filtering the first step of metagenomic data analysis requires the execution of certain pre-filtering steps, including the removal of redundant, low-quality sequences and sequences of probable eukaryotic origin (especially in metagenomes of human origin). the methods available for the removal of contaminating eukaryotic genomic dna sequences include eu-detect and deconseq. 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 highly non-redundant. 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. misassemblies 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 to 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. 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 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. after an assembly is created, an additional challenge is "metagenomic deconvolution", or determining which sequences come from which species in the sample. gene prediction metagenomic analysis pipelines use two approaches in the annotation of coding regions in the