A 4-lineage Statistical Suite to Evaluate the Support of Large-Scale Retrotransposon Insertion Data to Reconstruct Evolutionary Trees.

Retrophylogenomics makes use of genome-wide retrotransposon presence/absence insertion patterns to resolve questions in phylogeny and population genetics. In the genomics era, evaluating high-throughput data requires the associated development of appropriately powerful statistical tools. The currently used KKSC 3-lineage statistical test for estimating the significance of retrophylogenomic data is limited by the number of possible tree topologies it can assess in one step. To improve on this, we have extended the analysis to simultaneously compare four lineages, enabling us to evaluate ten distinct presence/absence insertion patterns for 26 possible tree topologies plus 129 trees with different incidences of hybridization or introgression. The new tool provides statistics for cases involving multiple ancestral hybridizations/introgressions, ancestral incomplete lineage sorting, bifurcation, and polytomy. The test is embedded in a user-friendly web R application (http://retrogenomics.uni-muenster.de:3838/hammlet/) and is available for use by the scientific community. [ancestral hybridization/introgression; ancestral incomplete lineage sorting (ILS); empirical distribution; KKSC-statistics; 4-lineage (4-LIN) insertion polymorphism; polytomy; retrophylogenomics.].

The multicomparative 2-n-way genome suite.

To effectively analyze the increasing amounts of available genomic data, improved comparative analytical tools that are accessible to and applicable by a broad scientific community are essential. We built the "2-n-way" software suite to provide a fundamental and innovative processing framework for revealing and comparing inserted elements among various genomes. The suite comprises two user-friendly web-based modules. The 2-way module generates pairwise whole-genome alignments of target and query species. The resulting genome coordinates of blocks (matching sequences) and gaps (missing sequences) from multiple 2-ways are then transferred to the n-way module and sorted into projects, in which user-defined coordinates from reference species are projected to the block/gap coordinates of orthologous loci in query species to provide comparative information about presence (blocks) or absence (gaps) patterns of targeted elements over many entire genomes and phylogroups. Thus, the 2-n-way software suite is ideal for performing multidirectional, non-ascertainment-biased screenings to extract all possible presence/absence data of user-relevant elements in orthologous sequences. To highlight its applicability and versatility, we used 2-n-way to expose approximately 100 lost introns in vertebrates, analyzed thousands of potential phylogenetically informative bat and whale retrotransposons, and novel human exons as well as thousands of human polymorphic retrotransposons.

Speciation network in Laurasiatheria: retrophylogenomic signals.

Rapid species radiation due to adaptive changes or occupation of new ecospaces challenges our understanding of ancestral speciation and the relationships of modern species. At the molecular level, rapid radiation with successive speciations over short time periods-too short to fix polymorphic alleles-is described as incomplete lineage sorting. Incomplete lineage sorting leads to random fixation of genetic markers and hence, random signals of relationships in phylogenetic reconstructions. The situation is further complicated when you consider that the genome is a mosaic of ancestral and modern incompletely sorted sequence blocks that leads to reconstructed affiliations to one or the other relative, depending on the fixation of their shared ancestral polymorphic alleles. The laurasiatherian relationships among Chiroptera, Perissodactyla, Cetartiodactyla, and Carnivora present a prime example for such enigmatic affiliations. We performed whole-genome screenings for phylogenetically diagnostic retrotransposon insertions involving the representatives bat (Chiroptera), horse (Perissodactyla), cow (Cetartiodactyla), and dog (Carnivora), and extracted among 162,000 preselected cases 102 virtually homoplasy-free, phylogenetically informative retroelements to draw a complete picture of the highly complex evolutionary relations within Laurasiatheria. All possible evolutionary scenarios received considerable retrotransposon support, leaving us with a network of affiliations. However, the Cetartiodactyla-Carnivora relationship as well as the basal position of Chiroptera and an ancestral laurasiatherian hybridization process did exhibit some very clear, distinct signals. The significant accordance of retrotransposon presence/absence patterns and flanking nucleotide changes suggest an important influence of mosaic genome structures in the reconstruction of species histories.

The genome of the vervet (Chlorocebus aethiops sabaeus).

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), for which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intra-generic phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population, we discover the first structural variations that are, in some cases, predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.

Incomplete Lineage Sorting and Hybridization Statistics for Large-Scale Retroposon Insertion Data.

Ancient retroposon insertions can be used as virtually homoplasy-free markers to reconstruct the phylogenetic history of species. Inherited, orthologous insertions in related species offer reliable signals of a common origin of the given species. One prerequisite for such a phylogenetically informative insertion is that the inserted element was fixed in the ancestral population before speciation; if not, polymorphically inserted elements may lead to random distributions of presence/absence states during speciation and possibly to apparently conflicting reconstructions of their ancestry. Fortunately, such misleading fixed cases are relatively rare but nevertheless, need to be considered. Here, we present novel, comprehensive statistical models applicable for (1) analyzing any pattern of rare genomic changes, (2) testing and differentiating conflicting phylogenetic reconstructions based on rare genomic changes caused by incomplete lineage sorting or/and ancestral hybridization, and (3) differentiating between search strategies involving genome information from one or several lineages. When the new statistics are applied, in non-conflicting cases a minimum of three elements present in both of two species and absent in a third group are considered significant support (p<0.05) for the branching of the third from the other two, if all three of the given species are screened equally for genome or experimental data. Five elements are necessary for significant support (p<0.05) if a diagnostic locus derived from only one of three species is screened, and no conflicting markers are detected. Most potentially conflicting patterns can be evaluated for their significance and ancestral hybridization can be distinguished from incomplete lineage sorting by considering symmetric or asymmetric distribution of rare genomic changes among possible tree configurations. Additionally, we provide an R-application to make the new KKSC insertion significance test available for the scientific community at http://retrogenomics.uni-muenster.de:3838/KKSC_significance_test/.

Exploring Massive Incomplete Lineage Sorting in Arctoids (Laurasiatheria, Carnivora).

Freed from the competition of large raptors, Paleocene carnivores could expand their newly acquired habitats in search of prey. Such changing conditions might have led to their successful distribution and rapid radiation. Today, molecular evolutionary biologists are faced, however, with the consequences of such accelerated adaptive radiations, because they led to sequential speciation more rapidly than phylogenetic markers could be fixed. The repercussions being that current genealogies based on such markers are incongruent with species trees.Our aim was to explore such conflicting phylogenetic zones of evolution during the early arctoid radiation, especially to distinguish diagnostic from misleading phylogenetic signals, and to examine other carnivore-related speciation events. We applied a combination of high-throughput computational strategies to screen carnivore and related genomes in silico for randomly inserted retroposed elements that we then used to identify inconsistent phylogenetic patterns in the Arctoidea group, which is well known for phylogenetic discordances.Our combined retrophylogenomic and in vitro wet lab approach detected hundreds of carnivore-specific insertions, many of them confirming well-established splits or identifying and solving conflicting species distributions. Our systematic genome-wide screens for Long INterspersed Elements detected homoplasy-free markers with insertion-specific truncation points that we used to distinguish phylogenetically informative markers from conflicting signals. The results were independently confirmed by phylogenetic diagnostic Short INterspersed Elements. As statistical analysis ruled out ancestral hybridization, these doubly verified but still conflicting patterns were statistically determined to be genomic remnants from a time of ancestral incomplete lineage sorting that especially accompanied large parts of Arctoidea evolution.

GPAC-genome presence/absence compiler: a web application to comparatively visualize multiple genome-level changes.

Our understanding of genome-wide and comparative sequence information has been broadened considerably by the databases available from the University of California Santa Cruz (UCSC) Genome Bioinformatics Department. In particular, the identification and visualization of genomic sequences, present in some species but absent in others, led to fundamental insights into gene and genome evolution. However, the UCSC tools currently enable one to visualize orthologous genomic loci for a range of species in only a single locus. For large-scale comparative analyses of such presence/absence patterns a multilocus view would be more desirable. Such a tool would enable us to compare thousands of relevant loci simultaneously and to resolve many different questions about, for example, phylogeny, specific aspects of genome and gene evolution, such as the gain or loss of exons and introns, the emergence of novel transposed elements, nonprotein-coding RNAs, and viral genomic particles. Here, we present the first tool to facilitate the parallel analysis of thousands of genomic loci for cross-species presence/absence patterns based on multiway genome alignments. This genome presence/absence compiler uses annotated or other compilations of coordinates of genomic locations and compiles all presence/absence patterns in a flexible, color-coded table linked to the individual UCSC Genome Browser alignments. We provide examples of the versatile information content of such a screening system especially for 7SL-derived transposed elements, nuclear mitochondrial DNA, DNA transposons, and miRNAs in primates (http://www.bioinformatics.uni-muenster.de/tools/gpac, last accessed October 1, 2014).

Ancestry of the Australian termitivorous numbat.

The Australian numbat, Myrmecobius fasciatus, is the only marsupial that feeds almost exclusively on termites and that has a life following the diurnally restricted and dynamic geographical distribution of termites. The millions of years of this adaptation led to unique morphological and anatomical features, especially basicranial and dental characteristics, that make it difficult to identify a clear phylogenetic affiliation to other marsupials. From DNA sequence analyses, the family Myrmecobiidae is placed within the dasyuromorph marsupials, but the exact position varies from study to study, and support values are mostly rather modest. Here, we report the recovery and analysis of approximately 110,000 quasifossilized traces of mobile element insertions into the genome of a dasyurid marsupial (Tasmanian devil), 25 of which are phylogenetically informative for early dasyuromorphial evolution. Fourteen of these ancient retroposon insertions are shared by the 16 Dasyuromorphia species analyzed, including the numbat, but are absent in the outgroups. An additional 11 other insertions are present in all Dasyuridae but are absent in the numbat. These findings place numbats as the sister group to all living Dasyuridae and show that the investigated Dasyuromorphia, including the Myrmecobiidae, constitutes a monophyletic group that is separated from Peramelemorphia, Notoryctemorphia, and other marsupials.

Retroposon insertion patterns of neoavian birds: strong evidence for an extensive incomplete lineage sorting era.

More than 150 Ma, the avian lineage separated from that of other dinosaurs and later diversified into the more than 10,000 species extant today. The early neoavian bird radiations most likely occurred in the late Cretaceous (more than 65 Ma) but left behind few if any molecular signals of their archaic evolutionary past. Retroposed elements, once established in an ancestral population, are highly valuable, virtually homoplasy-free markers of species evolution; after applying stringent orthology criteria, their phylogenetically informative presence/absence patterns are free of random noise and independent of evolutionary rate or nucleotide composition effects. We screened for early neoavian orthologous retroposon insertions and identified six markers with conflicting presence/absence patterns, whereas six additional retroposons established before or after the presumed major neoavian radiation show consistent phylogenetic patterns. The exceptionally frequent conflicting retroposon presence/absence patterns of neoavian orders are strong indicators of an extensive incomplete lineage sorting era, potentially induced by an early rapid successive speciation of ancestral Neoaves.

Tracking marsupial evolution using archaic genomic retroposon insertions.

The Australasian and South American marsupial mammals, such as kangaroos and opossums, are the closest living relatives to placental mammals, having shared a common ancestor around 130 million years ago. The evolutionary relationships among the seven marsupial orders have, however, so far eluded resolution. In particular, the relationships between the four Australasian and three South American marsupial orders have been intensively debated since the South American order Microbiotheria was taxonomically moved into the group Australidelphia. Australidelphia is significantly supported by both molecular and morphological data and comprises the four Australasian marsupial orders and the South American order Microbiotheria, indicating a complex, ancient, biogeographic history of marsupials. However, the exact phylogenetic position of Microbiotheria within Australidelphia has yet to be resolved using either sequence or morphological data analysis. Here, we provide evidence from newly established and virtually homoplasy-free retroposon insertion markers for the basal relationships among marsupial orders. Fifty-three phylogenetically informative markers were retrieved after in silico and experimental screening of approximately 217,000 retroposon-containing loci from opossum and kangaroo. The four Australasian orders share a single origin with Microbiotheria as their closest sister group, supporting a clear divergence between South American and Australasian marsupials. In addition, the new data place the South American opossums (Didelphimorphia) as the first branch of the marsupial tree. The exhaustive computational and experimental evidence provides important insight into the evolution of retroposable elements in the marsupial genome. Placing the retroposon insertion pattern in a paleobiogeographic context indicates a single marsupial migration from South America to Australia. The now firmly established phylogeny can be used to determine the direction of genomic changes and morphological transitions within marsupials.

Euarchontoglires Challenged by Incomplete Lineage Sorting.

Euarchontoglires, once described as Supraprimates, comprise primates, colugos, tree shrews, rodents, and lagomorphs in a clade that evolved about 90 million years ago (mya) from a shared ancestor with Laurasiatheria. The rapid speciation of groups within Euarchontoglires, and the subsequent inherent incomplete marker fixation in ancestral lineages, led to challenged attempts at phylogenetic reconstructions, particularly for the phylogenetic position of tree shrews. To resolve this conundrum, we sampled genome-wide presence/absence patterns of transposed elements (TEs) from all representatives of Euarchontoglires. This specific marker system has the advantage that phylogenetic diagnostic characters can be extracted in a nearly unbiased fashion genome-wide from reference genomes. Their insertions are virtually free of homoplasy. We simultaneously employed two computational tools, the genome presence/absence compiler (GPAC) and 2-n-way, to find a maximum of diagnostic insertions from more than 3 million TE positions. From 361 extracted diagnostic TEs, 132 provide significant support for the current resolution of Primatomorpha (Primates plus Dermoptera), 94 support the union of Euarchonta (Primates, Dermoptera, plus Scandentia), and 135 marker insertion patterns support a variety of alternative phylogenetic scenarios. Thus, whole genome-level analysis and a virtually homoplasy-free marker system offer an opportunity to finally resolve the notorious phylogenetic challenges that nature produces in rapidly diversifying groups.

Rodent evolution: back to the root.

Some 70 Ma, rodents arose along a branch of our own mammalian lineage. Today, about 40% of all mammalian species are rodents and are found in vast numbers on almost every continent. Not only is their proliferation extensive but also the rates of DNA evolution vary significantly among lineages, which has hindered attempts to reconstruct, especially the root of, their evolutionary history. The presence or absence of rare genomic changes, such as short interspersed elements (SINEs), are, however, independent of high molecular substitution rates and provide a powerful, virtually homoplasy-free source for solving such phylogenetic problems. We screened 12 Gb of rodent genomic information using whole-genome three-way alignments, multiple lineage-specific sequences, high-throughput polymerase chain reaction amplifications, and sequencing to reveal 65 phylogenetically informative SINE insertions dispersed over 23 rodent phylogenetic nodes. Eight SINEs and six indels provide significant support for an early association of the Mouse-related and Ctenohystrica (guinea pig and relatives) clades, the Squirrel-related clade being the sister group. This early speciation scenario was also evident in the genomewide distribution pattern of B1-related retroposons, as mouse and guinea pig genomes share six such retroposon subfamilies, containing hundreds of thousands of elements that are clearly absent in the ground squirrel genome. Interestingly, however, two SINE insertions and one diagnostic indel support an association of Ctenohystrica with the Squirrel-related clade. Lineage sorting or a more complex evolutionary scenario that includes an early divergence of the Squirrel-related ancestor and a subsequent hybridization of the latter and the Ctenohystrica lineage best explains such apparently contradictory insertions.

Retroposon insertions provide insights into deep lagomorph evolution.

The homogenous mammalian order Lagomorpha comprises about 80 species in two families, Ochotonidae (pikas) and Leporidae (rabbits and hares). However, the phylogenetic relationships among leporids are controversial. Molecular data, particularly from mitochondrial sequences, give highly homoplasious signals. To resolve the controversy between mitochondrial and nuclear data, we analyzed genomic orthologous retroposon insertion sites, a virtually homoplasy-free marker system. From a differential screen of rabbit genomic data for intronic retroposon insertions of CSINE elements, we polymerase chain reaction-amplified and sequenced 11 retroposons in eight representative lagomorphs. We found three retroposons shared among all lagomorphs but absent in outgroups, four confirmed the monophyly of leporids, and three significantly supported Pronolagus as the sister group to all other leporids. One retroposon supported the monophyly of Lepus. The position of Pronolagus outside of the remaining leporids supports the sequence-based signals of nuclear genes and clearly refutes the misleading signals of mitochondrial genes.

A novel web-based TinT application and the chronology of the Primate Alu retroposon activity.

BACKGROUND: DNA sequences afford access to the evolutionary pathways of life. Particularly mobile elements that constantly co-evolve in genomes encrypt recent and ancient information of their host's history. In mammals there is an extraordinarily abundant activity of mobile elements that occurs in a dynamic succession of active families, subfamilies, types, and subtypes of retroposed elements. The high frequency of retroposons in mammals implies that, by chance, such elements also insert into each other. While inactive elements are no longer able to retropose, active elements retropose by chance into other active and inactive elements. Thousands of such directional, element-in-element insertions are found in present-day genomes. To help analyze these events, we developed a computational algorithm (Transpositions in Transpositions, or TinT) that examines the different frequencies of nested transpositions and reconstructs the chronological order of retroposon activities. RESULTS: By examining the different frequencies of such nested transpositions, the TinT application reconstructs the chronological order of retroposon activities. We use such activity patterns as a comparative tool to (1) delineate the historical rise and fall of retroposons and their relations to each other, (2) understand the retroposon-induced complexity of recent genomes, and (3) find selective informative homoplasy-free markers of phylogeny. The efficiency of the new application is demonstrated by applying it to dimeric Alu Short INterspersed Elements (SINE) to derive a complete chronology of such elements in primates. CONCLUSION: The user-friendly, web-based TinT interface presented here affords an easy, automated screening for nested transpositions from genome assemblies or trace data, assembles them in a frequency-matrix, and schematically displays their chronological activity history.

Evolutionary history of 7SL RNA-derived SINEs in Supraprimates.

The evolutionary relationships of 7SL RNA-derived SINEs such as the primate Alu or the rodent B1 elements have hitherto been obscure. We established an unambiguous phylogenetic tree for Supraprimates, and derived intraordinal relationships of the 7SL RNA-derived SINEs. As well as new elements in Tupaia and primates, we also found that the purported ancestral fossil Alu monomer was restricted to Primates, and provide here the first description of a potential chimeric promoter box region in SINEs.

Retroposed elements as archives for the evolutionary history of placental mammals.

Reconstruction of the placental mammalian (eutherian) evolutionary tree has undergone diverse revisions, and numerous aspects remain hotly debated. Initial hierarchical divisions based on morphology contained many misgroupings due to features that evolved independently by similar selection processes. Molecular analyses corrected many of these misgroupings and the superordinal hierarchy of placental mammals was recently assembled into four clades. However, long or rapid evolutionary periods, as well as directional mutation pressure, can produce molecular homoplasies, similar characteristics lacking common ancestors. Retroposed elements, by contrast, integrate randomly into genomes with negligible probabilities of the same element integrating independently into orthologous positions in different species. Thus, presence/absence analyses of these elements are a superior strategy for molecular systematics. By computationally scanning more than 160,000 chromosomal loci and judiciously selecting from only phylogenetically informative retroposons for experimental high-throughput PCR applications, we recovered 28 clear, independent monophyly markers that conclusively verify the earliest divergences in placental mammalian evolution. Using tests that take into account ancestral polymorphisms, multiple long interspersed elements and long terminal repeat element insertions provide highly significant evidence for the monophyletic clades Boreotheria (synonymous with Boreoeutheria), Supraprimates (synonymous with Euarchontoglires), and Laurasiatheria. More importantly, two retropositions provide new support for a prior scenario of early mammalian evolution that places the basal placental divergence between Xenarthra and Epitheria, the latter comprising all remaining placentals. Due to its virtually homoplasy-free nature, the analysis of retroposon presence/absence patterns avoids the pitfalls of other molecular methodologies and provides a rapid, unequivocal means for revealing the evolutionary history of organisms.

The Volcano Rabbit in the Phylogenetic Network of Lagomorphs.

The order Lagomorpha unifies pikas (Ochotonidae) and the hares plus rabbits (Leporidae). Phylogenetic reconstructions of the species within Leporidae based on traditional morphological or molecular sequence data provide support for conflicting hypotheses. The retroposon presence/absence patterns analyzed in this study revealed strong support for the broadly accepted splitting of lagomorphs into ochotonids and leporids with Pronolagus as the first divergence in the leporid tree. Furthermore, the retroposon presence/absence patterns nested the rare volcano rabbit, Romerolagus diazi, within an unresolved network of deeper leporid relationships and provide the first homoplasy-free image of incomplete lineage sorting and/or ancestral hybridization/introgression in rapidly radiated Leporidae. At the same time, the strongest retroposon presence/absence signal supports the volcano rabbit as a separate branch between the Pronolagus junction and a unified cluster of the remaining leporids.

Waves of genomic hitchhikers shed light on the evolution of gamebirds (Aves: Galliformes).

BACKGROUND: The phylogenetic tree of Galliformes (gamebirds, including megapodes, currassows, guinea fowl, New and Old World quails, chicken, pheasants, grouse, and turkeys) has been considerably remodeled over the last decades as new data and analytical methods became available. Analyzing presence/absence patterns of retroposed elements avoids the problems of homoplastic characters inherent in other methodologies. In gamebirds, chicken repeats 1 (CR1) are the most prevalent retroposed elements, but little is known about the activity of their various subtypes over time. Ascertaining the fixation patterns of CR1 elements would help unravel the phylogeny of gamebirds and other poorly resolved avian clades. RESULTS: We analyzed 1,978 nested CR1 elements and developed a multidimensional approach taking advantage of their transposition in transposition character (TinT) to characterize the fixation patterns of all 22 known chicken CR1 subtypes. The presence/absence patterns of those elements that were active at different periods of gamebird evolution provided evidence for a clade (Cracidae + (Numididae + (Odontophoridae + Phasianidae))) not including Megapodiidae; and for Rollulus as the sister taxon of the other analyzed Phasianidae. Genomic trace sequences of the turkey genome further demonstrated that the endangered African Congo Peafowl (Afropavo congensis) is the sister taxon of the Asian Peafowl (Pavo), rejecting other predominantly morphology-based groupings, and that phasianids are monophyletic, including the sister taxa Tetraoninae and Meleagridinae. CONCLUSION: The TinT information concerning relative fixation times of CR1 subtypes enabled us to efficiently investigate gamebird phylogeny and to reconstruct an unambiguous tree topology. This method should provide a useful tool for investigations in other taxonomic groups as well.

The Beaver's Phylogenetic Lineage Illuminated by Retroposon Reads.

Solving problematic phylogenetic relationships often requires high quality genome data. However, for many organisms such data are still not available. Among rodents, the phylogenetic position of the beaver has always attracted special interest. The arrangement of the beaver's masseter (jaw-closer) muscle once suggested a strong affinity to some sciurid rodents (e.g., squirrels), placing them in the Sciuromorpha suborder. Modern molecular data, however, suggested a closer relationship of beaver to the representatives of the mouse-related clade, but significant data from virtually homoplasy-free markers (for example retroposon insertions) for the exact position of the beaver have not been available. We derived a gross genome assembly from deposited genomic Illumina paired-end reads and extracted thousands of potential phylogenetically informative retroposon markers using the new bioinformatics coordinate extractor fastCOEX, enabling us to evaluate different hypotheses for the phylogenetic position of the beaver. Comparative results provided significant support for a clear relationship between beavers (Castoridae) and kangaroo rat-related species (Geomyoidea) (p < 0.0015, six markers, no conflicting data) within a significantly supported mouse-related clade (including Myodonta, Anomaluromorpha, and Castorimorpha) (p < 0.0015, six markers, no conflicting data).

RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria.

Recent bioinformatics-aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot-gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs co-expressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III-dependent cleavage. To the best of our knowledge, this represents the first case of two non-coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half-lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed.