The Role of DNA Methylation in Genome Defense in Cnidaria and Other Invertebrates.

Considerable attention has recently been focused on the potential involvement of DNA methylation in regulating gene expression in cnidarians. Much of this work has been centered on corals, in the context of changes in methylation perhaps facilitating adaptation to higher seawater temperatures and other stressful conditions. Although first proposed more than 30 years ago, the possibility that DNA methylation systems function in protecting animal genomes against the harmful effects of transposon activity has largely been ignored since that time. Here, we show that transposons are specifically targeted by the DNA methylation system in cnidarians, and that the youngest transposons (i.e., those most likely to be active) are most highly methylated. Transposons in longer and highly active genes were preferentially methylated and, as transposons aged, methylation levels declined, reducing the potentially harmful side effects of CpG methylation. In Cnidaria and a range of other invertebrates, correlation between the overall extent of methylation and transposon content was strongly supported. Present transposon burden is the dominant factor in determining overall level of genomic methylation in a range of animals that diverged in or before the early Cambrian, suggesting that genome defense represents the ancestral role of CpG methylation.

Conservation and turnover of miRNAs and their highly complementary targets in early branching animals.

MicroRNAs (miRNAs) are crucial post-transcriptional regulators that have been extensively studied in Bilateria, a group comprising the majority of extant animals, where more than 30 conserved miRNA families have been identified. By contrast, bilaterian miRNA targets are largely not conserved. Cnidaria is the sister group to Bilateria and thus provides a unique opportunity for comparative studies. Strikingly, like their plant counterparts, cnidarian miRNAs have been shown to predominantly have highly complementary targets leading to transcript cleavage by Argonaute proteins. Here, we assess the conservation of miRNAs and their targets by small RNA sequencing followed by miRNA target prediction in eight species of Anthozoa (sea anemones and corals), the earliest-branching cnidarian class. We uncover dozens of novel miRNAs but only a few conserved ones. Further, given their high complementarity, we were able to computationally identify miRNA targets in each species. Besides evidence for conservation of specific miRNA target sites, which are maintained between sea anemones and stony corals across 500 Myr of evolution, we also find indications for convergent evolution of target regulation by different miRNAs. Our data indicate that cnidarians have only few conserved miRNAs and corresponding targets, despite their high complementarity, suggesting a high evolutionary turnover.

Expression of the neuropeptides RFamide and LWamide during development of the coral Acropora millepora in relation to settlement and metamorphosis.

Neuropeptides play critical roles in cnidarian development. However, although they are known to play key roles in settlement and metamorphosis, their temporal and spatial developmental expression has not previously been characterized in any coral. We here describe Acropora millepora LWamide and RFamide and their developmental expression from the time of their first appearance, using in situ hybridization and FMRFamide immunohistochemistry. AmRFamide transcripts first appear in the ectoderm toward the oral end of the planula larva following blastopore closure. This oral bias becomes less apparent as the planula develops. The cell bodies of AmRFamide-expressing cells are centrally located in the ectoderm, with narrow projections to the mesoglea and to the cell surface. As the planula approaches settlement, AmRFamide expression disappears and is undetectable in the newly settled polyp. Expressing cells then gradually reappear as the polyp develops, becoming particularly abundant on the tentacles. AmLWamide transcripts first appear in ectodermal cells of the developing planula, with minimal expression at its two ends. The cell bodies of expressing cells lie just above the mesoglea, in a position distinct from those of AmRFamide-expressing cells, and have a narrow projection extending across the ectoderm to its surface. AmLWamide-expressing cells persist for most of the planula stage, disappearing shortly before settlement, but later than AmRFamide-expressing cells. As is the case with AmRFamide, expressing cells are absent from the polyp immediately after settlement, reappearing later on its oral side. AmLWamide expression lags that of AmRFamide in both its disappearance and reappearance. Antibodies to FMRFamide stain cells in a pattern similar to that of the transcripts, but also cells in areas where there is no expression revealed by in situ hybridization, most notably at the aboral end of the planula and in the adult polyp. Adult polyps have numerous staining cells on the tentacles and oral discs, as well as an immunoreactive nerve ring around the mouth. There are scattered staining cells in the coenosarc between polyps and staining cells are abundant in the mesenterial filaments. The above results are discussed in the context of our knowledge of the behavior of coral planulae at the time of their settlement and metamorphosis. Corals are facing multiple environmental threats, and these results both highlight the need for, and bring us a step closer to, a mechanistic understanding of a process that is critical to their survival.

Host modification of a bacterial quorum-sensing signal induces a phenotypic switch in bacterial symbionts.

Bacterial communities colonize epithelial surfaces of most animals. Several factors, including the innate immune system, mucus composition, and diet, have been identified as determinants of host-associated bacterial communities. Here we show that the early branching metazoan Hydra is able to modify bacterial quorum-sensing signals. We identified a eukaryotic mechanism that enables Hydra to specifically modify long-chain 3-oxo-homoserine lactones into their 3-hydroxy-HSL counterparts. Expression data revealed that Hydra's main bacterial colonizer, Curvibacter sp., responds differentially to N-(3-hydroxydodecanoyl)-l-homoserine lactone (3OHC12-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). Investigating the impacts of the different N-acyl-HSLs on host colonization elucidated that 3OHC12-HSL allows and 3OC12-HSL represses host colonization of Curvibacter sp. These results show that an animal manipulates bacterial quorum-sensing signals and that this modification leads to a phenotypic switch in the bacterial colonizers. This mechanism may enable the host to manipulate the gene expression and thereby the behavior of its bacterial colonizers.

Transcriptomic analysis reveals protein homeostasis breakdown in the coral Acropora millepora during hypo-saline stress.

BACKGROUND: Coral reefs can experience salinity fluctuations due to rainfall and runoff; these events can have major impacts on the corals and lead to bleaching and mortality. On the Great Barrier Reef (GBR), low salinity events, which occur during summer seasons and can involve salinity dropping ~ 10 PSU correlate with declines in coral cover, and these events are predicted to increase in frequency and severity under future climate change scenarios. In other marine invertebrates, exposure to low salinity causes increased expression of genes involved in proteolysis, responses to oxidative stress, and membrane transport, but the effects that changes in salinity have on corals have so far received only limited attention. To better understand the coral response to hypo-osmotic stress, here we investigated the transcriptomic response of the coral Acropora millepora in both adult and juvenile life stages to acute (1 h) and more prolonged (24 h) exposure to low salinity. RESULTS: Differential gene expression analysis revealed the involvement of both common and specific response mechanisms in Acropora. The general response to environmental stressors included up-regulation of genes involved in the mitigation of macromolecular and oxidative damage, while up-regulation of genes involved in amino acid metabolism and transport represent specific responses to salinity stress. CONCLUSIONS: This study is the first comprehensive transcriptomic analysis of the coral response to low salinity stress and provides important insights into the likely consequences of heavy rainfall and runoff events on coral reefs.

DMSP biosynthesis by an animal and its role in coral thermal stress response.

Globally, reef-building corals are the most prolific producers of dimethylsulphoniopropionate (DMSP), a central molecule in the marine sulphur cycle and precursor of the climate-active gas dimethylsulphide. At present, DMSP production by corals is attributed entirely to their algal endosymbiont, Symbiodinium. Combining chemical, genomic and molecular approaches, we show that coral juveniles produce DMSP in the absence of algal symbionts. DMSP levels increased up to 54% over time in newly settled coral juveniles lacking algal endosymbionts, and further increases, up to 76%, were recorded when juveniles were subjected to thermal stress. We uncovered coral orthologues of two algal genes recently identified in DMSP biosynthesis, strongly indicating that corals possess the enzymatic machinery necessary for DMSP production. Our results overturn the paradigm that photosynthetic organisms are the sole biological source of DMSP, and highlight the double jeopardy represented by worldwide declining coral cover, as the potential to alleviate thermal stress through coral-produced DMSP declines correspondingly.

Transcriptomic analysis of the response of Acropora millepora to hypo-osmotic stress provides insights into DMSP biosynthesis by corals.

BACKGROUND: Dimethylsulfoniopropionate (DMSP) is a small sulphur compound which is produced in prodigious amounts in the oceans and plays a pivotal role in the marine sulfur cycle. Until recently, DMSP was believed to be synthesized exclusively by photosynthetic organisms; however we now know that corals and specific bacteria can also produce this compound. Corals are major sources of DMSP, but the molecular basis for its biosynthesis is unknown in these organisms. RESULTS: Here we used salinity stress, which is known to trigger DMSP production in other organisms, in conjunction with transcriptomics to identify coral genes likely to be involved in DMSP biosynthesis. We focused specifically on both adults and juveniles of the coral Acropora millepora: after 24 h of exposure to hyposaline conditions, DMSP concentrations increased significantly by 2.6 fold in adult corals and 1.2 fold in juveniles. Concomitantly, candidate genes enabling each of the necessary steps leading to DMSP production were up-regulated. CONCLUSIONS: The data presented strongly suggest that corals use an algal-like pathway to generate DMSP from methionine, and are able to rapidly change expression of the corresponding genes in response to environmental stress. However, our data also indicate that DMSP is unlikely to function primarily as an osmolyte in corals, instead potentially serving as a scavenger of ROS and as a molecular sink for excess methionine produced as a consequence of proteolysis and osmolyte catabolism in corals under hypo-osmotic conditions.

A comparative view of early development in the corals Favia lizardensis, Ctenactis echinata, and Acropora millepora - morphology, transcriptome, and developmental gene expression.

BACKGROUND: Research into various aspects of coral biology has greatly increased in recent years due to anthropogenic threats to coral health including pollution, ocean warming and acidification. However, knowledge of coral early development has lagged. The present paper describes the embryonic development of two previously uncharacterized robust corals, Favia lizardensis (a massive brain coral) and Ctenactis echinata (a solitary coral) and compares it to that of the previously characterized complex coral, Acropora millepora, both morphologically and in terms of the expression of a set of key developmental genes. RESULTS: Illumina sequencing of mixed age embryos was carried out, resulting in embryonic transcriptomes consisting of 40605 contigs for C.echinata (N50 = 1080 bp) and 48536 contigs for F.lizardensis (N50 = 1496 bp). The transcriptomes have been annotated against Swiss-Prot and were sufficiently complete to enable the identification of orthologs of many key genes controlling development in bilaterians. Developmental series of images of whole mounts and sections reveal that the early stages of both species contain a blastocoel, consistent with their membership of the robust clade. In situ hybridization was used to examine the expression of the developmentally important genes brachyury, chordin and forkhead. The expression of brachyury and forkhead was consistent with that previously reported for Acropora and allowed us to confirm that the pseudo-blastopore sometimes seen in robust corals such as Favia spp. is not directly associated with gastrulation. C.echinata chordin expression, however, differed from that seen in the other two corals. CONCLUSIONS: Embryonic transcriptomes were assembled for the brain coral Favia lizardensis and the solitary coral Ctenactis echinata. Both species have a blastocoel in their early developmental stages, consistent with their phylogenetic position as members of the robust clade. Expression of the key developmental genes brachyury, chordin and forkhead was investigated, allowing comparison to that of their orthologs in Acropora, Nematostella and bilaterians and demonstrating that even within the Anthozoa there are significant differences in expression patterns.

Functional conservation of the apoptotic machinery from coral to man: the diverse and complex Bcl-2 and caspase repertoires of Acropora millepora.

BACKGROUND: Apoptotic cell death is a defining and ubiquitous characteristic of metazoans, but its evolutionary origins are unclear. Although Caenorhabditis and Drosophila played key roles in establishing the molecular bases of apoptosis, it is now clear that cell death pathways of these animals do not reflect ancestral characteristics. Conversely, recent work suggests that the apoptotic networks of cnidarians may be complex and vertebrate-like, hence characterization of the apoptotic complement of representatives of the basal cnidarian class Anthozoa will help us to understand the evolution of the vertebrate apoptotic network. RESULTS: We describe the Bcl-2 and caspase protein repertoires of the coral Acropora millepora, making use of the comprehensive transcriptomic data available for this species. Molecular phylogenetics indicates that some Acropora proteins are orthologs of specific mammalian pro-apoptotic Bcl-2 family members, but the relationships of other Bcl-2 and caspases are unclear. The pro- or anti-apoptotic activities of coral Bcl-2 proteins were investigated by expression in mammalian cells, and the results imply functional conservation of the effector/anti-apoptotic machinery despite limited sequence conservation in the anti-apoptotic Bcl-2 proteins. A novel caspase type ("Caspase-X"), containing both inactive and active caspase domains, was identified in Acropora and appears to be restricted to corals. When expressed in mammalian cells, full-length caspase-X caused loss of viability, and a truncated version containing only the active domain was more effective in inducing cell death, suggesting that the inactive domain might modulate activity in the full-length protein. Structure prediction suggests that the active and inactive caspase domains in caspase-X are likely to interact, resulting in a structure resembling that of the active domain in procaspase-8 and the inactive caspase domain in the mammalian c-FLIP anti-apoptotic factor. CONCLUSIONS: The data presented here confirm that many of the basic mechanisms involved in both the intrinsic and extrinsic apoptotic pathways were in place in the common ancestor of cnidarians and bilaterians. With the identification of most or all of the repertoires of coral Bcl-2 and caspases, our results not only provide new perspectives on the evolution of apoptotic pathways, but also a framework for future experimental studies towards a complete understanding of coral bleaching mechanisms, in which apoptotic cell death might be involved.

Hologenome analysis of two marine sponges with different microbiomes.

BACKGROUND: Sponges (Porifera) harbor distinct microbial consortia within their mesohyl interior. We herein analysed the hologenomes of Stylissa carteri and Xestospongia testudinaria, which notably differ in their microbiome content. RESULTS: Our analysis revealed that S. carteri has an expanded repertoire of immunological domains, specifically Scavenger Receptor Cysteine-Rich (SRCR)-like domains, compared to X. testudinaria. On the microbial side, metatranscriptome analyses revealed an overrepresentation of potential symbiosis-related domains in X. testudinaria. CONCLUSIONS: Our findings provide genomic insights into the molecular mechanisms underlying host-symbiont coevolution and may serve as a roadmap for future hologenome analyses.

Near-future pH conditions severely impact calcification, metabolism and the nervous system in the pteropod Heliconoides inflatus.

Shelled pteropods play key roles in the global carbon cycle and food webs of various ecosystems. Their thin external shell is sensitive to small changes in pH, and shell dissolution has already been observed in areas where aragonite saturation state is ~1. A decline in pteropod abundance has the potential to disrupt trophic networks and directly impact commercial fisheries. Therefore, it is crucial to understand how pteropods will be affected by global environmental change, particularly ocean acidification. In this study, physiological and molecular approaches were used to investigate the response of the Mediterranean pteropod, Heliconoides inflatus, to pH values projected for 2100 under a moderate emissions trajectory (RCP6.0). Pteropods were subjected to pHT 7.9 for 3 days, and gene expression levels, calcification and respiration rates were measured relative to pHT 8.1 controls. Gross calcification decreased markedly under low pH conditions, while genes potentially involved in calcification were up-regulated, reflecting the inability of pteropods to maintain calcification rates. Gene expression data imply that under low pH conditions, both metabolic processes and protein synthesis may be compromised, while genes involved in acid-base regulation were up-regulated. A large number of genes related to nervous system structure and function were also up-regulated in the low pH treatment, including a GABAA receptor subunit. This observation is particularly interesting because GABAA receptor disturbances, leading to altered behavior, have been documented in several other marine animals after exposure to elevated CO2 . The up-regulation of many genes involved in nervous system function suggests that exposure to low pH could have major effects on pteropod behavior. This study illustrates the power of combining physiological and molecular approaches. It also reveals the importance of behavioral analyses in studies aimed at understanding the impacts of low pH on marine animals.

How do environmental factors influence life cycles and development? An experimental framework for early-diverging metazoans.

Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans. Also watch the Video Abstract.

Estimation of the methylation pattern distribution from deep sequencing data.

BACKGROUND: Bisulphite sequencing enables the detection of cytosine methylation. The sequence of the methylation states of cytosines on any given read forms a methylation pattern that carries substantially more information than merely studying the average methylation level at individual positions. In order to understand better the complexity of DNA methylation landscapes in biological samples, it is important to study the diversity of these methylation patterns. However, the accurate quantification of methylation patterns is subject to sequencing errors and spurious signals due to incomplete bisulphite conversion of cytosines. RESULTS: A statistical model is developed which accounts for the distribution of DNA methylation patterns at any given locus. The model incorporates the effects of sequencing errors and spurious reads, and enables estimation of the true underlying distribution of methylation patterns. CONCLUSIONS: Calculation of the estimated distribution over methylation patterns is implemented in the R Bioconductor package MPFE. Source code and documentation of the package are also available for download at http://bioconductor.org/packages/3.0/bioc/html/MPFE.html .

DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees.

In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non-protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

The acute transcriptional response of the coral Acropora millepora to immune challenge: expression of GiMAP/IAN genes links the innate immune responses of corals with those of mammals and plants.

BACKGROUND: As a step towards understanding coral immunity we present the first whole transcriptome analysis of the acute responses of Acropora millepora to challenge with the bacterial cell wall derivative MDP and the viral mimic poly I:C, defined immunogens provoking distinct but well characterised responses in higher animals. RESULTS: These experiments reveal similarities with the responses both of arthropods and mammals, as well as coral-specific effects. The most surprising finding was that MDP specifically induced three members of the GiMAP gene family, which has been implicated in immunity in mammals but is absent from Drosophila and Caenorhabditis. Like their mammalian homologs, GiMAP genes are arranged in a tandem cluster in the coral genome. CONCLUSIONS: A phylogenomic survey of this gene family implies ancient origins, multiple independent losses and lineage-specific expansions during animal evolution. Whilst functional convergence cannot be ruled out, GiMAP expression in corals may reflect an ancestral role in immunity, perhaps in phagolysosomal processing.

New tricks with old genes: the genetic bases of novel cnidarian traits.

Recent thought on genome evolution has focused on the creation of new genes and changes in regulatory mechanisms while ignoring the role of selective gene loss in shaping genomes. Using data from two cnidarians, the jellyfish Clytia and the coral Acropora, we examined the relative significance of new 'taxonomically restricted' genes and selectively retained ancestral genes in enabling the evolution of novel traits. Consistent with its more complex life-cycle, the proportion of novel genes identified in Clytia was higher than that in the 'polyp only' cnidarians Nematostella and Hydra, but each of these cnidarians has retained a proportion of ancestral genes not present in the other two. The ubiquity and near-stochastic nature of gene loss can explain the discord between patterns of gene distribution and taxonomy.

The honey bee epigenomes: differential methylation of brain DNA in queens and workers.

In honey bees (Apis mellifera) the behaviorally and reproductively distinct queen and worker female castes derive from the same genome as a result of differential intake of royal jelly and are implemented in concert with DNA methylation. To determine if these very different diet-controlled phenotypes correlate with unique brain methylomes, we conducted a study to determine the methyl cytosine (mC) distribution in the brains of queens and workers at single-base-pair resolution using shotgun bisulfite sequencing technology. The whole-genome sequencing was validated by deep 454 sequencing of selected amplicons representing eight methylated genes. We found that nearly all mCs are located in CpG dinucleotides in the exons of 5,854 genes showing greater sequence conservation than non-methylated genes. Over 550 genes show significant methylation differences between queens and workers, revealing the intricate dynamics of methylation patterns. The distinctiveness of the differentially methylated genes is underscored by their intermediate CpG densities relative to drastically CpG-depleted methylated genes and to CpG-richer non-methylated genes. We find a strong correlation between methylation patterns and splicing sites including those that have the potential to generate alternative exons. We validate our genome-wide analyses by a detailed examination of two transcript variants encoded by one of the differentially methylated genes. The link between methylation and splicing is further supported by the differential methylation of genes belonging to the histone gene family. We propose that modulation of alternative splicing is one mechanism by which DNA methylation could be linked to gene regulation in the honey bee. Our study describes a level of molecular diversity previously unknown in honey bees that might be important for generating phenotypic flexibility not only during development but also in the adult post-mitotic brain.

Phylogenomics reveals an anomalous distribution of USP genes in metazoans.

Members of the universal stress protein (USP) family were originally identified in stressed bacteria on the basis of a shared domain, which has since been reported in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants. Although not previously characterized in metazoans, here we report that USP genes are distributed in animal genomes in a unique pattern that reflects frequent independent losses and independent expansions. Multiple USP loci are present in urochordates as well as all Cnidaria and Lophotrochozoa examined, but none were detected in any of the available ecdysozoan or non-urochordate deuterostome genome data. The vast majority of the metazoan USPs are short, single-domain proteins and are phylogenetically distinct from the prokaryotic, plant, protist, and fungal members of the protein family. Whereas most of the metazoan USP genes contain introns, with few exceptions those in the cnidarian Hydra are intronless and cluster together in phylogenetic analyses. Expression patterns were determined for several cnidarian USPs, including two genes belonging to the intronless clade, and these imply diverse functions. The apparent paradox of implied diversity of roles despite high overall levels of sequence (and implied structural) similarity parallels the situation in bacteria. The absence of USP genes in ecdysozoans and most deuterostomes may be a consequence of functional redundancy or specialization in taxon-specific roles.

Morphological stasis masks ecologically divergent coral species on tropical reefs.

Coral reefs are the epitome of species diversity, yet the number of described scleractinian coral species, the framework-builders of coral reefs, remains moderate by comparison. DNA sequencing studies are rapidly challenging this notion by exposing a wealth of undescribed diversity, but the evolutionary and ecological significance of this diversity remains largely unclear. Here, we present an annotated genome for one of the most ubiquitous corals in the Indo-Pacific (Pachyseris speciosa) and uncover, through a comprehensive genomic and phenotypic assessment, that it comprises morphologically indistinguishable but ecologically divergent lineages. Demographic modeling based on whole-genome resequencing indicated that morphological crypsis (across micro- and macromorphological traits) was due to ancient morphological stasis rather than recent divergence. Although the lineages occur sympatrically across shallow and mesophotic habitats, extensive genotyping using a rapid molecular assay revealed differentiation of their ecological distributions. Leveraging "common garden" conditions facilitated by the overlapping distributions, we assessed physiological and quantitative skeletal traits and demonstrated concurrent phenotypic differentiation. Lastly, spawning observations of genotyped colonies highlighted the potential role of temporal reproductive isolation in the limited admixture, with consistent genomic signatures in genes related to morphogenesis and reproduction. Overall, our findings demonstrate the presence of ecologically and phenotypically divergent coral species without substantial morphological differentiation and provide new leads into the potential mechanisms facilitating such divergence. More broadly, they indicate that our current taxonomic framework for reef-building corals may be scratching the surface of the ecologically relevant diversity on coral reefs, consequently limiting our ability to protect or restore this diversity effectively.

Characterizing the D2 statistic: word matches in biological sequences.

Word matches are often used in sequence comparison methods, either as a measure of sequence similarity or in the first search steps of algorithms such as BLAST or BLAT. The D2 statistic is the number of matches of words of k letters between two sequences. Recent advances have been made in the characterization of this statistic and in the approximation of its distribution. Here, these results are extended to the case of approximate word matches. We compute the exact value of the variance of the D2 statistic for the case of a uniform letter distribution, and introduce a method to provide accurate approximations of the variance in the remaining cases. This enables the distribution of D2 to be approximated for typical situations arising in biological research. We apply these results to the identification of cis-regulatory modules, and show that this method detects such sequences with a high accuracy. The ability to approximate the distribution of D2 for both exact and approximate word matches will enable the use of this statistic in a more precise manner for sequence comparison, database searches, and identification of transcription factor binding sites.