htsget: a protocol for securely streaming genomic data.

Summary: Standardized interfaces for efficiently accessing high-throughput sequencing data are a fundamental requirement for large-scale genomic data sharing. We have developed htsget, a protocol for secure, efficient and reliable access to sequencing read and variation data. We demonstrate four independent client and server implementations, and the results of a comprehensive interoperability demonstration. Availability and implementation: http://samtools.github.io/hts-specs/htsget.html. Supplementary information: Supplementary data are available at Bioinformatics online.

Alternative splicing and the evolution of phenotypic novelty.

Alternative splicing, a mechanism of post-transcriptional RNA processing whereby a single gene can encode multiple distinct transcripts, has been proposed to underlie morphological innovations in multicellular organisms. Genes with developmental functions are enriched for alternative splicing events, suggestive of a contribution of alternative splicing to developmental programmes. The role of alternative splicing as a source of transcript diversification has previously been compared to that of gene duplication, with the relationship between the two extensively explored. Alternative splicing is reduced following gene duplication with the retention of duplicate copies higher for genes which were alternatively spliced prior to duplication. Furthermore, and unlike the case for overall gene number, the proportion of alternatively spliced genes has also increased in line with the evolutionary diversification of cell types, suggesting alternative splicing may contribute to the complexity of developmental programmes. Together these observations suggest a prominent role for alternative splicing as a source of functional innovation. However, it is unknown whether the proliferation of alternative splicing events indeed reflects a functional expansion of the transcriptome or instead results from weaker selection acting on larger species, which tend to have a higher number of cell types and lower population sizes.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Alternative Splice in Alternative Lice.

Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation.

Correcting for differential transcript coverage reveals a strong relationship between alternative splicing and organism complexity.

What at the genomic level underlies organism complexity? Although several genomic features have been associated with organism complexity, in the case of alternative splicing, which has long been proposed to explain the variation in complexity, no such link has been established. Here, we analyzed over 39 million expressed sequence tags available for 47 eukaryotic species with fully sequenced genomes to obtain a comparable index of alternative splicing estimates, which corrects for the distorting effect of a variable number of transcripts per species--an important obstacle for comparative studies of alternative splicing. We find that alternative splicing has steadily increased over the last 1,400 My of eukaryotic evolution and is strongly associated with organism complexity, assayed as the number of cell types. Importantly, this association is not explained as a by-product of covariance between alternative splicing with other variables previously linked to complexity including gene content, protein length, proteome disorder, and protein interactivity. In addition, we found no evidence to suggest that the relationship of alternative splicing to cell type number is explained by drift due to reduced N(e) in more complex species. Taken together, our results firmly establish alternative splicing as a significant predictor of organism complexity and are, in principle, consistent with an important role of transcript diversification through alternative splicing as a means of determining a genome's functional information capacity.

Presence-absence variation in A. thaliana is primarily associated with genomic signatures consistent with relaxed selective constraints.

The sequencing of multiple genomes of the same plant species has revealed polymorphic gene and exon loss. Genes associated with disease resistance are overrepresented among those showing structural variations, suggesting an adaptive role for gene and exon presence-absence variation (PAV). To shed light on the possible functional relevance of polymorphic coding region loss and the mechanisms driving this process, we characterized genes that have lost entire exons or their whole coding regions in 17 fully sequenced Arabidopsis thaliana accessions. We found that although a significant enrichment in genes associated with certain functional categories is observed, PAV events are largely restricted to genes with signatures of reduced essentiality: PAV genes tend to be newer additions to the genome, tissue specific, and lowly expressed. In addition, PAV genes are located in regions of lower gene density and higher transposable element density. Partial coding region PAV events were associated with only a marginal reduction in gene expression level in the affected accession and occurred in genes with higher levels of alternative splicing in the Col-0 accession. Together, these results suggest that although adaptive scenarios cannot be ruled out, PAV events can be explained without invoking them.

Evidence for deep phylogenetic conservation of exonic splice-related constraints: splice-related skews at exonic ends in the brown alga Ectocarpus are common and resemble those seen in humans.

The control of RNA splicing is often modulated by exonic motifs near splice sites. Chief among these are exonic splice enhancers (ESEs). Well-described ESEs in mammals are purine rich and cause predictable skews in codon and amino acid usage toward exonic ends. Looking across species, those with relatively abundant intronic sequence are those with the more profound end of exon skews, indicative of exonization of splice site recognition. To date, the only intron-rich species that have been analyzed are mammals, precluding any conclusions about the likely ancestral condition. Here, we examine the patterns of codon and amino acid usage in the vicinity of exon-intron junctions in the brown alga Ectocarpus siliculosus, a species with abundant large introns, known SR proteins, and classical splice sites. We find that amino acids and codons preferred/avoided at both 3' and 5' ends in Ectocarpus, of which there are many, tend, on average, to also be preferred/avoided at the same exon ends in humans. Moreover, the preferences observed at the 5' ends of exons are largely the same as those at the 3' ends, a symmetry trend only previously observed in animals. We predict putative hexameric ESEs in Ectocarpus and show that these are purine rich and that there are many more of these identified as functional ESEs in humans than expected by chance. These results are consistent with deep phylogenetic conservation of SR protein binding motifs. Assuming codons preferred near boundaries are "splice optimal" codons, in Ectocarpus, unlike Drosophila, splice optimal and translationally optimal codons are not mutually exclusive. The exclusivity of translationally optimal and splice optimal codon sets is thus not universal.

Alternative splicing: a potential source of functional innovation in the eukaryotic genome.

Alternative splicing (AS) is a common posttranscriptional process in eukaryotic organisms, by which multiple distinct functional transcripts are produced from a single gene. The release of the human genome draft revealed a much smaller number of genes than anticipated. Because of its potential role in expanding protein diversity, interest in alternative splicing has been increasing over the last decade. Although recent studies have shown that 94% human multiexon genes undergo AS, evolution of AS and thus its potential role in functional innovation in eukaryotic genomes remain largely unexplored. Here we review available evidence regarding the evolution of AS prevalence and functional role. In addition we stress the need to correct for the strong effect of transcript coverage in AS detection and set out a strategy to ultimately elucidate the extent of the role of AS in functional innovation on a genomic scale.

Increased levels of noisy splicing in cancers, but not for oncogene-derived transcripts.

Recent genome-wide analyses have detected numerous cancer-specific alternative splicing (AS) events. Whether transcripts containing cancer-specific AS events are likely to be translated into functional proteins or simply reflect noisy splicing, thereby determining their clinical relevance, is not known. Here we show that consistent with a noisy-splicing model, cancer-specific AS events generally tend to be rare, containing more premature stop codons and have less identifiable functional domains in both the human and mouse. Interestingly, common cancer-derived AS transcripts from tumour suppressor and oncogenes show marked changes in premature stop-codon frequency; with tumour suppressor genes exhibiting increased levels of premature stop codons whereas oncogenes have the opposite pattern. We conclude that tumours tend to have faithful oncogene splicing and a higher incidence of premature stop codons among tumour suppressor and cancer-specific splice variants showing the importance of considering splicing noise when analysing cancer-specific splicing changes.