Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor

BACKGROUND: Repbase is a reference database of eukaryotic repetitive DNA, which includes prototypic sequences of repeats and basic information described in annotations. Updating and maintenance of the database requires specialized tools, which we have created and made available for use with Repbase, and which may be useful as a template for other curated databases. RESULTS: We describe the software tools RepbaseSubmitter and Censor, which are designed to facilitate updating and screening the content of Repbase. RepbaseSubmitter is a java-based interface for formatting and annotating Repbase entries. It eliminates many common formatting errors, and automates actions such as calculation of sequence lengths and composition, thus facilitating curation of Repbase sequences. In addition, it has several features for predicting protein coding regions in sequences; searching and including Pubmed references in Repbase entries; and searching the NCBI taxonomy database for correct inclusion of species information and taxonomic position. Censor is a tool to rapidly identify repetitive elements by comparison to known repeats. It uses WU-BLAST for speed and sensitivity, and can conduct DNA-DNA, DNA-protein, or translated DNA-translated DNA searches of genomic sequence. Defragmented output includes a map of repeats present in the query sequence, with the options to report masked query sequence(s), repeat sequences found in the query, and alignments. CONCLUSION: Censor and RepbaseSubmitter are available as both web-based services and downloadable versions. They can be found at (RepbaseSubmitter) and (Censor)

Attempts to detect retrotransposition and de novo deletion of Alus and other dispersed repeats at specific loci in the human genome

Dispersed repeat elements contribute to genome instability by de novo insertion and unequal recombination between repeats. To study the dynamics of these processes, we have developed single DNA molecule approaches to detect de novo insertions at a single locus and Alu-mediated deletions at two different loci in human genomic DNA. Validation experiments showed these approaches could detect insertions and deletions at frequencies below 10(-6) per cell. However, bulk analysis of germline (sperm) and somatic DNA showed no evidence for genuine mutant molecules, placing an upper limit of insertion and deletion rates of 2 x 10(-7) and 3 x 10(-7), respectively, in the individuals tested. Such re-arrangements at these loci therefore occur at a rate lower than that detectable by the most sensitive methods currently available

Analysis of the human Alu Ye lineage

Background: Alu elements are short (∼300 bp) interspersed elements that amplify in primate genomes through a process termed retroposition. The expansion of these elements has had a significant impact on the structure and function of primate genomes. Approximately 10 % of the mass of the human genome is comprised of Alu elements, making them the most abundant short interspersed element (SINE) in our genome. The majority of Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that occurred 30-50 million years ago. Alu elements are therefore a rich source of inter- and intra-species primate genomic variation. Results: A total of 153 Alu elements from the Ye subfamily were extracted from the draft sequence of the human genome. Analysis of these elements resulted in the discovery of two new Alu subfamilies, Ye4 and Ye6, complementing the previously described Ye5 subfamily. DNA sequence analysis of each of the Alu Ye subfamilies yielded average age estimates of ∼14, ∼13 and ∼9.5 million years old for the Alu Ye4, Ye5 and Ye6 subfamilies, respectively. In addition, 120 Alu Ye4, Ye5 and Ye6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity. Conclusion: The Alu Ye lineage appears to have started amplifying relatively early in primate evolution and continued propagating at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Detailed sequence analysis of several Alu pre-integration sites indicated that multiple types of events had occurred, including gene conversions, near-parallel independent insertions of different Alu elements and Alu-mediated genomic deletions. A potential hotspot for Alu insertion in the Fer1L3 gene on chromosome 10 was also identified. © 2005 Salem et al; licensee BioMed Central Ltd

The diversity of Class II transposable elements in mammalian genomes has arisen from ancestral phylogenetic splits during ancient waves of proliferation through the genome

DNA transposons make up three percent of the human genome, roughly the same percentage as genes. However, due to their inactivity, they are often ignored in favour of the more abundant, active, retroelements. Despite this relative ignominy, there are a number of interesting questions to be asked of these transposon families. One particular question relates to the timing of proliferation and inactivation of elements in a family. Does an ongoing process of turnover occur, or is the process more akin to a life cycle for the family, with elements proliferating rapidly before deactivation at a later date? We answer this question by tracing back to the most recent common ancestor of each modern transposon family, using two different methods. The first method identifies the most recent common ancestor of the species in which a family of transposon fossils can still be found, which we assume will have existed soon after the true origin date of the transposon family. The second method uses molecular dating techniques to predict the age of the most recent common ancestor element from which all elements found in a modern genome are descended. Independent data from five pairs of species are used in the molecular dating analysis: Human- Chimpanzee, Human-Orangutan, Dog-Panda, Dog-Cat and Cow-Pig. Orthologous pairs of elements from host species pairs are included, and the divergence dates of these species are used to constrain the analysis. We discover that, in general, the times to element common ancestry, for a given family, are the same for the different species pairs, suggesting that there has been no order-specific process of turnover. Furthermore, for most families, the ages of the common ancestor of the host species and of that of the elements are similar, suggesting a life cycle model for the proliferation of transposons. Where these two ages differ, in families found only in Primates and Rodentia, for example, we find that the host species date is later than that of the common ancestor of the elements, implying that there may be large deletions of elements from host species, examples of which were found in their ancestors

Genomic repeat abundances contain phylogenetic signal

A large proportion of genomic information, particularly repetitive elements, is usually ignored when researchers are using next-generation sequencing. Here we demonstrate the usefulness of this repetitive fraction in phylogenetic analyses, utilizing comparative graph-based clustering of next-generation sequence reads, which results in abundance estimates of different classes of genomic repeats. Phylogenetic trees are then inferred based on the genome-wide abundance of different repeat types treated as continuously varying characters; such repeats are scattered across chromosomes and in angiosperms can constitute a majority of nuclear genomic DNA. In six diverse examples, five angiosperms and one insect, this method provides generally well-supported relationships at interspecific and intergeneric levels that agree with results from more standard phylogenetic analyses of commonly used markers. We propose that this methodology may prove especially useful in groups where there is little genetic differentiation in standard phylogenetic markers. At the same time as providing data for phylogenetic inference, this method additionally yields a wealth of data for comparative studies of genome evolution

Repetitive Elements May Comprise Over Two-Thirds of the Human Genome

Transposable elements (TEs) are conventionally identified in eukaryotic genomes by alignment to consensus element sequences. Using this approach, about half of the human genome has been previously identified as TEs and low-complexity repeats. We recently developed a highly sensitive alternative de novo strategy, P-clouds, that instead searches for clusters of high-abundance oligonucleotides that are related in sequence space (oligo “clouds”). We show here that P-clouds predicts >840 Mbp of additional repetitive sequences in the human genome, thus suggesting that 66%–69% of the human genome is repetitive or repeat-derived. To investigate this remarkable difference, we conducted detailed analyses of the ability of both P-clouds and a commonly used conventional approach, RepeatMasker (RM), to detect different sized fragments of the highly abundant human Alu and MIR SINEs. RM can have surprisingly low sensitivity for even moderately long fragments, in contrast to P-clouds, which has good sensitivity down to small fragment sizes (∼25 bp). Although short fragments have a high intrinsic probability of being false positives, we performed a probabilistic annotation that reflects this fact. We further developed “element-specific” P-clouds (ESPs) to identify novel Alu and MIR SINE elements, and using it we identified ∼100 Mb of previously unannotated human elements. ESP estimates of new MIR sequences are in good agreement with RM-based predictions of the amount that RM missed. These results highlight the need for combined, probabilistic genome annotation approaches and suggest that the human genome consists of substantially more repetitive sequence than previously believed

SINEs, evolution and genome structure in the opossum

Short INterspersed Elements (SINEs) are non-autonomous retrotransposons, usually between 100 and 500 base pairs (bp) in length, which are ubiquitous components of eukaryotic genomes. Their activity, distribution, and evolution can be highly informative on genomic structure and evolutionary processes. To determine recent activity, we amplified more than one hundred SINE1 loci in a panel of 43 M. domestica individuals derived from five diverse geographic locations. The SINE1 family has expanded recently enough that many loci were polymorphic, and the SINE1 insertion-based genetic distances among populations reflected geographic distance. Genome-wide comparisons of SINE1 densities and GC content revealed that high SINE1 density is associated with high GC content in a few long and many short spans. Young SINE1s, whether fixed or polymorphic, showed an unbiased GC content preference for insertion, indicating that the GC preference accumulates over long time periods, possibly in periodic bursts. SINE1 evolution is thus broadly similar to human Alu evolution, although it has an independent origin. High GC content adjacent to SINE1s is strongly correlated with bias towards higher AT to GC substitutions and lower GC to AT substitutions. This is consistent with biased gene conversion, and also indicates that like chickens, but unlike eutherian mammals, GC content heterogeneity (isochore structure) is reinforced by substitution processes in the M. domestica genome. Nevertheless, both high and low GC content regions are apparently headed towards lower GC content equilibria, possibly due to a relative shift to lower recombination rates in the recent Monodelphis ancestral lineage. Like eutherians, metatherian (marsupial) mammals have evolved high CpG substitution rates, but this is apparently a convergence in process rather than a shared ancestral state. © 2007 Elsevier B.V. All rights reserved

Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome

Transposable elements (TEs) have no longer been totally considered as “junk DNA” for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C(chromosomeconformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r=0.9, P<2.2×1016; IMR90 fibroblasts: r = 0.94, P < 2.2 × 1016) and also have a significant positive correlation withsomeremote functional DNA elements like enhancers and promoters (Enhancer: hESC: r=0.997, P=2.3×10−4; IMR90: r=0.934, P=2×10−2; Promoter: hESC: r = 0.995, P = 3.8 × 10−4; IMR90: r = 0.996, P = 3.2 × 10−4). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes