Search

Summary: The program tuple_plot identifies and visualizes local similarities between two genomic sequences, typically 100 kb or longer, by applying the well-known dotplot principle. A dictionary of sequence words built from the input sequences serves to construct a task-specific expectancy model that is used to attribute significance values to pairwise word hits. The dictionary-based approach allows fast computation, the computation time scaling to O(N log N), depending on the size of the input sequences. The proposed scoring scheme appreciably increases the signal-to-noise ratio and may help to improve other word-based sequence comparison approaches., Availability: tuple_plot is available at http://genome.fli-leibniz.de/software.html and may be used under GNU public license.

Background: In highly copy number variable (CNV) regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS) approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach. Results: As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations. Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs) including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods. Conclusion: Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation. [ABSTRACT FROM AUTHOR]

Background: Subtle alternative splicing events involving tandem splice sites separated by a short (2-12 nucleotides) distance are frequent and evolutionarily widespread in eukaryotes, and a major contributor to the complexity of transcriptomes and proteomes. However, these events have been either omitted altogether in databases on alternative splicing, or only the cases of experimentally confirmed alternative splicing have been reported. Thus, a database which covers all confirmed cases of subtle alternative splicing as well as the numerous putative tandem splice sites (which might be confirmed once more transcript data becomes available), and allows to search for tandem splice sites with specific features and download the results, is a valuable resource for targeted experimental studies and large-scale bioinformatics analyses of tandem splice sites. Towards this goal we recently set up TassDB (Tandem Splice Site DataBase, version 1), which stores data about alternative splicing events at tandem splice sites separated by 3 nt in eight species. Description: We have substantially revised and extended TassDB. The currently available version 2 contains extensive information about tandem splice sites separated by 2-12 nt for the human and mouse transcriptomes including data on the conservation of the tandem motifs in five vertebrates. TassDB2 offers a user-friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download result datasets. For example, users can search for cases of alternative splicing where the proportion of EST/mRNA evidence supporting the minor isoform exceeds a specific threshold, or where the difference in splice site scores is specified by the user. The predicted impact of each event on the protein is also reported, along with information about being a putative target for the nonsense-mediated decay (NMD) pathway. Links are provided to the UCSC genome browser and other external resources. Conclusion: TassDB2, available via http://www.tassdb.info, provides comprehensive resources for researchers interested in both targeted experimental studies and large-scale bioinformatics analyses of short distance tandem splice sites. [ABSTRACT FROM AUTHOR]

Background: Alternative selection of splice sites in tandem donors and acceptors is a major mode of alternative splicing. Here, we analyzed whether in-frame tandem sites leading to subtle mRNA insertions/deletions of 3, 6, or 9 nucleotides are under natural selection. Results: We found multiple lines of evidence that the human protein coding sequences are under selection against such in-frame tandem splice events, indicating that these events are often deleterious. The strength of selection is not homogeneous within the coding sequence as protein regions that fold into a fixed 3D structure (intrinsically ordered) are under stronger selection, especially against sites with a strong minor splice site. Investigating structures of functional protein domains, we found that tandem acceptors are preferentially located at the domain surface and outside structural elements such as helices and sheets. Using three-species comparisons, we estimate that more than half of all mutations that create NAGNAG acceptors in the coding region have been eliminated by selection. Conclusion: We estimate that ~2,400 introns are under selection against possessing a tandem site. [ABSTRACT FROM AUTHOR]

Background: The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels of alternative tandem splice site exons occurring in different human organ systems and cell types, and to study known sequence features involved in the recognition and distinction of neighboring splice sites, we performed large-scale, stringent alignments of cDNA sequences and ESTs to the human and mouse genomes, followed by experimental validation. Results: We analyzed alternative 5'ss exons (A5Es) and alternative 3'ss exons (A3Es), derived from transcript sequences that were aligned to assembled genome sequences to infer patterns of AS occurring in several thousands of genes. Comparing the levels of overlapping (tandem) and non-overlapping (competitive) A5Es and A3Es, a clear preference of isoforms was seen for tandem acceptors and donors, with four nucleotides and three to six nucleotides long exon extensions, respectively. A subset of inferred A5E tandem exons was selected and experimentally validated. With the focus on A5Es, we investigated their transcript coverage, sequence conservation and base-paring to U1 snRNA, proximal and distal splice site classification, candidate motifs for cis-regulatory activity, and compared A5Es with A3Es, constitutive and pseudo-exons, in H. sapiens and M. musculus. The results reveal a small but authentic enriched set of tandem splice site preference, with specific distances between proximal and distal 5'ss (3'ss), which showed a marked dichotomy between the levels of in- and out-of-frame splicing for A5Es and A3Es, respectively, identified a number of candidate NMD targets, and allowed a rough estimation of a number of undetected tandem donors based on splice site information. Conclusion: This comparative study distinguishes tandem 5'ss and 3'ss, with three to six nucleotides long extensions, as having unusually high proportions of AS, experimentally validates tandem donors in a panel of different human tissues, highlights the dichotomy in the types of AS occurring at tandem splice sites, and elucidates that human alternative exons spliced at overlapping 5'ss posses features of typical splice variants that could well be beneficial for the cell. [ABSTRACT FROM AUTHOR]

Several species have genomes in which the four nucleotides are not equally represented (Glöckner 2000). Interestingly, shifts to very high A/T or G/C levels can occur in several distinct branches of the tree of life. The underlying reasons for these shifts therefore may be of different origin. Now entire chromosome sequences from two different A/T-rich genomes,Dictyostelium discoideumandPlasmodium falciparum, are available (Bowman et al. 1999; Gardner et al. 2002; Glöckner et al. 2002). This gives us the opportunity to investigate how a high A/T content may influence the signals that are the landmarks for gene specification. We found that, in contrast with most known metazoan and plant genomes, splice signals contain, little information other than the canonical GT-AG dinucleotides. Intron lengths in A/T rich organisms, on the other hand, are comparable to those of other lower eukaryotes. Intergenic regions show, dependent on the orientation of adjacent genes, a size pattern with a ratio of 1 (3'-3') to 2 (3'-5') to 3 (5'-5'). Overall, gene organization patterns seem not to be influenced by the A/T bias. Surprisingly, the slightly higher A/T content of theP. falciparumgenome compared to that ofD. discoideum(80.1 versus 77.4%) is not achieved by increased A/T richness in intergenic regions. Instead both the shift of the nucleotide usage in coding regions to A/T-rich codons and the longer intergenic regions make an equal contribution to the higher A/T content in this organism. [ABSTRACT FROM AUTHOR]