Genome Research

Detecting copy number variation with mated short reads.

Publication Date: 2010 Aug 30 PMID: 20805290
Authors: Medvedev, P. - Fiume, M. - Dzamba, M. - Smith, T. - Brudno, M.
Journal: Genome Res

The development of high-throughput sequencing (HTS) technologies has opened the door to novel methods for detecting copy number variants (CNVs) in the human genome. While in the past CNVs have been detected based on array CGH data, recent studies have shown that depth-of-coverage information from HTS technologies can also be used for the reliable identification of large copy-variable regions. Such methods, however, are hindered by sequencing biases that lead certain regions of the genome to be over- or under- sampled, lowering their resolution and ability to accurately identify the exact breakpoints of the variants. In this work, we develop a method for CNV detection that supplements the depth-of-coverage with paired-end mapping information, where matepairs mapping discordantly to the reference serve to indicate the presence of variation. Our algorithm, called CNVer, combines this information within a unified computational framework called the donor graph, allowing us to better mitigate the sequencing biases that cause uneven local coverage and accurately predict CNVs. We use CNVer to detect 4879 CNVs in the recently described genome of a Yoruban individual. Most of the calls (77%) coincide with previously known variants within the Database of Genomic Variants, while 81% of deletion copy number variants previously known for this individual coincide with one of our loss calls. Furthermore, we demonstrate that CNVer can reconstruct the absolute copy counts of segments of the donor genome and evaluate the feasibility of using CNVer with low coverage datasets.

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Computational analysis of genome-wide DNA methylation during the differentiation of human embryonic stem cells along the endodermal lineage.

Publication Date: 2010 Aug 27 PMID: 20802089
Authors: Chavez, L. - Jozefczuk, J. - Grimm, C. - Dietrich, J. - Timmermann, B. - Lehrach, H. - Herwig, R. - Adjaye, J.
Journal: Genome Res

The generation of genome-wide data derived from methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) has become a major tool for epigenetic studies in health and disease. The computational analysis of such data, however, still falls short on accuracy, sensitivity, and speed. We propose a time-efficient statistical method that is able to cope with the inherent complexity of MeDIP-seq data with similar performance compared with existing methods. In order to demonstrate the computational approach, we have analyzed alterations in DNA methylation during the differentiation of human embryonic stem cells (hESCs) to definitive endoderm. We show improved correlation of normalized MeDIP-seq data in comparison to available whole-genome bisulfite sequencing data, and investigated the effect of differential methylation on gene expression. Furthermore, we analyzed the interplay between DNA methylation, histone modifications, and transcription factor binding and show that in contrast to de novo methylation, demethylation is mainly associated with regions of low CpG densities.

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Age-dependent chromosomal distribution of male-biased genes in Drosophila.

Publication Date: 2010 Aug 26 PMID: 20798392
Authors: Zhang, Y. E. - Vibranovski, M. D. - Krinsky, B. H. - Long, M.
Journal: Genome Res

We investigated the correlation between the chromosomal location and age distribution of new male-biased genes formed by duplications via DNA intermediates (DNA-level) or by de novo origination in Drosophila. Our genome-wide analysis revealed an excess of young X-linked male-biased genes. The proportion of X-linked male-biased genes then diminishes through time leading to an autosomal excess of male-biased genes. The switch between X-linked and autosomal enrichment of male-biased genes was also present in the distribution of both protein-coding genes on the D. pseudoobscura neo-X chromosome and microRNA genes of D. melanogaster. These observations revealed that the evolution of male-biased genes is more complicated than the previously detected one-step X->A gene traffic and the enrichment of the male-biased genes on autosomes. The pattern we detected suggests that the interaction of various evolutionary forces such as the meiotic sex chromosome inactivation (MSCI), faster-X effect, and sexual antagonism in the male germline might have shaped the chromosomal distribution of male-biased genes on different evolutionary time scales.

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A ChIP-seq defined genome-wide map of vitamin D receptor binding: Associations with disease and evolution.

Publication Date: 2010 Aug 24 PMID: 20736230
Authors: Ramagopalan, S. V. - Heger, A. - Berlanga, A. J. - Maugeri, N. J. - Lincoln, M. R. - Burrell, A. - Handunnetthi, L. - Handel, A. E. - Disanto, G. - Orton, S. M. - Watson, C. T. - Morahan, J. M. - Giovannoni, G. - Ponting, C. P. - Ebers, G. C. - Knight, J. C.
Journal: Genome Res

Initially thought to play a restricted role in calcium homeostasis, the pleiotropic actions of vitamin D in biology and their clinical significance are only now becoming apparent. However, the mode of action of vitamin D, through its cognate nuclear vitamin D receptor (VDR), and its contribution to diverse disorders, remain poorly understood. We determined VDR binding throughout the human genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After calcitriol stimulation, we identified 2776 genomic positions occupied by the VDR and 229 genes with significant changes in expression in response to vitamin D. VDR binding sites were significantly enriched near autoimmune and cancer associated genes identified from genome-wide association (GWA) studies. Notable genes with VDR binding included IRF8, associated with MS, and PTPN2 associated with Crohn's disease and T1D. Furthermore, a number of single nucleotide polymorphism associations from GWA were located directly within VDR binding intervals, for example, rs13385731 associated with SLE and rs947474 associated with T1D. We also observed significant enrichment of VDR intervals within regions of positive selection among individuals of Asian and European descent. ChIP-seq determination of transcription factor binding, in combination with GWA data, provides a powerful approach to further understanding the molecular bases of complex diseases.

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Reshaping the gut microbiome with bacterial transplantation and antibiotic intake.

Publication Date: 2010 Aug 24 PMID: 20736229
Authors: Manichanh, C. - Reeder, J. - Gibert, P. - Varela, E. - Llopis, M. - Antolin, M. - Guigo, R. - Knight, R. - Guarner, F.
Journal: Genome Res

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.

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A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

Publication Date: 2010 Aug 18 PMID: 20719920
Authors: Burroughs, A. M. - Ando, Y. - de Hoon, M. J. - Tomaru, Y. - Nishibu, T. - Ukekawa, R. - Funakoshi, T. - Kurokawa, T. - Suzuki, H. - Hayashizaki, Y. - Daub, C. O.
Journal: Genome Res

Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

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Genome architecture marked by retrotransposons modulates predisposition to DNA methylation in cancer.

Publication Date: 2010 Aug 17 PMID: 20716667
Authors: Estecio, M. R. - Gallegos, J. - Vallot, C. - Castoro, R. J. - Chung, W. - Maegawa, S. - Oki, Y. - Kondo, Y. - Jelinek, J. - Shen, L. - Hartung, H. - Aplan, P. D. - Czerniak, B. A. - Liang, S. - Issa, J. P.
Journal: Genome Res

Epigenetic silencing plays an important role in cancer development. An attractive hypothesis is that local DNA features may participate in differential predisposition to gene hypermethylation. We found that, compared with methylation-resistant genes, methylation-prone genes have a lower frequency of SINE and LINE retrotransposons near their transcription start site. In several large testing sets, this distribution was highly predictive of promoter methylation. Genome-wide analysis showed that 22% of human genes were predicted to be methylation-prone in cancer; these tended to be genes that are down-regulated in cancer and that function in developmental processes. Moreover, retrotransposon distribution marks a larger fraction of methylation-prone genes compared to Polycomb group protein (PcG) marking in embryonic stem cells; indeed, PcG marking and our predictive model based on retrotransposon frequency appear to be correlated but also complementary. In summary, our data indicate that retrotransposon elements, which are widespread in our genome, are strongly associated with gene promoter DNA methylation in cancer and may in fact play a role in influencing epigenetic regulation in normal and abnormal physiological states.

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p53 binds preferentially to genomic regions with high DNA-encoded nucleosome occupancy.

Publication Date: 2010 Aug 17 PMID: 20716666
Authors: Lidor Nili, E. - Field, Y. - Lubling, Y. - Widom, J. - Oren, M. - Segal, E.
Journal: Genome Res

The human transcription factor TP53 is a pivotal roadblock against cancer. A key unresolved question is how the p53 protein selects its genomic binding sites in vivo out of a large pool of potential consensus sites. We hypothesized that chromatin may play a significant role in this site-selection process. To test this, we used a custom DNA microarray to measure p53 binding at approximately 2000 sites predicted to possess high-sequence specificity, and identified both strongly bound and weakly bound sites. When placed within a plasmid, weakly bound sites become p53 responsive and regain p53 binding when stably integrated into random genomic locations. Notably, strongly bound sites reside preferentially within genomic regions whose DNA sequence is predicted to encode relatively high intrinsic nucleosome occupancy. Using in vivo nucleosome occupancy measurements under conditions where p53 is inactive, we experimentally confirmed this prediction. Furthermore, upon p53 activation, nucleosomes are partially displaced from a relatively broad region surrounding the bound p53 sites, and this displacement is rapidly reversed upon inactivation of p53. Thus, in contrast to the general assumption that transcription-factor binding is preferred in sites that have low nucleosome occupancy prior to factor activation, we find that p53 binding occurs preferentially within a chromatin context of high intrinsic nucleosome occupancy.

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Changes in H2A.Z occupancy and DNA methylation during B-cell lymphomagenesis.

Publication Date: 2010 Aug 13 PMID: 20709945
Authors: Conerly, M. L. - Teves, S. S. - Diolaiti, D. - Ulrich, M. - Eisenman, R. N. - Henikoff, S.
Journal: Genome Res

The histone variant H2A.Z has been implicated in the regulation of gene expression, and in plants antagonizes DNA methylation. Here, we ask whether a similar relationship exists in mammals, using a mouse B-cell lymphoma model, where chromatin states can be monitored during tumorigenesis. Using native chromatin immunoprecipitation with microarray hybridization (ChIP-chip), we found a progressive depletion of H2A.Z around transcriptional start sites (TSSs) during MYC-induced transformation of pre-B cells and, subsequently, during lymphomagenesis. In addition, we found that H2A.Z and DNA methylation are generally anticorrelated around TSSs in both wild-type and MYC-transformed cells, as expected for the opposite effects of these chromatin features on promoter competence. Depletion of H2A.Z over TSSs both in cells that are induced to proliferate and in cells that are developing into a tumor suggests that progressive loss of H2A.Z during tumorigenesis results from the advancing disease state. These changes were accompanied by increases in chromatin salt solubility. Surprisingly, approximately 30% of all genes showed a redistribution of H2A.Z from around TSSs to bodies of active genes during the transition from MYC-transformed to tumor cells, with DNA methylation lost from gene bodies where H2A.Z levels increased. No such redistributions were observed during MYC-induced transformation of wild-type pre-B cells. The documented role of H2A.Z in regulating transcription suggests that 30% of genes have the potential to be aberrantly expressed during tumorigenesis. Our results imply that antagonism between H2A.Z deposition and DNA methylation is a conserved feature of eukaryotic genes, and that transcription-coupled H2A.Z changes may play a role in cancer initiation and progression.

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Signatures of positive selection apparent in a small sample of human exomes.

Publication Date: 2010 Aug 30 PMID: 20693481
Authors: Tennessen, J. A. - Madeoy, J. - Akey, J. M.
Journal: Genome Res

Exome sequences, which comprise all protein-coding regions, are promising data sets for studies of natural selection because they offer unbiased genome-wide estimates of polymorphism while focusing on the portions of the genome that are most likely to be functionally important. We examine genomic patterns of polymorphism within 10 diploid autosomal exomes of European and African descent. Using coalescent simulations, we show how polymorphism, site frequency spectra, and intercontinental divergence in these samples would be influenced by different modes of positive selection. We examine putatively selected loci from four previous genome-wide scans of SNP genotypes and demonstrate that these regions indeed show unusual population genetic patterns in the exome data. Using a series of conservative criteria based on exome polymorphism, we are able to fine-scale map signatures of selection, in many cases pinpointing a single candidate SNP. We also identify and evaluate novel candidate selection genes that show unusual patterns of polymorphism. We sequence a portion of one novel candidate locus, IVL, in 74 individuals from multiple continents and examine global genetic diversity. Thus, we confirm, narrow, and supplement existing catalogs of putative targets of selection, and show that exome data sets, which are likely to soon become common, will be powerful tools for identifying adaptive genetic variation.

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Massive turnover of functional sequence in human and other mammalian genomes.

Publication Date: 2010 Sep 1 PMID: 20693480
Authors: Meader, S. - Ponting, C. P. - Lunter, G.
Journal: Genome Res

Despite the availability of dozens of animal genome sequences, two key questions remain unanswered: First, what fraction of any species' genome confers biological function, and second, are apparent differences in organismal complexity reflected in an objective measure of genomic complexity? Here, we address both questions by applying, across the mammalian phylogeny, an evolutionary model that estimates the amount of functional DNA that is shared between two species' genomes. Our main findings are, first, that as the divergence between mammalian species increases, the predicted amount of pairwise shared functional sequence drops off dramatically. We show by simulations that this is not an artifact of the method, but rather indicates that functional (and mostly noncoding) sequence is turning over at a very high rate. We estimate that between 200 and 300 Mb ( approximately 6.5%-10%) of the human genome is under functional constraint, which includes five to eight times as many constrained noncoding bases than bases that code for protein. In contrast, in D. melanogaster we estimate only 56-66 Mb to be constrained, implying a ratio of noncoding to coding constrained bases of about 2. This suggests that, rather than genome size or protein-coding gene complement, it is the number of functional bases that might best mirror our naive preconceptions of organismal complexity.

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Optimization of de novo transcriptome assembly from next-generation sequencing data.

Publication Date: 2010 Aug 30 PMID: 20693479
Authors: Surget-Groba, Y. - Montoya-Burgos, J. I.
Journal: Genome Res

Transcriptome analysis has important applications in many biological fields. However, assembling a transcriptome without a known reference remains a challenging task requiring algorithmic improvements. We present two methods for substantially improving transcriptome de novo assembly. The first method relies on the observation that the use of a single k-mer length by current de novo assemblers is suboptimal to assemble transcriptomes where the sequence coverage of transcripts is highly heterogeneous. We present the Multiple-k method in which various k-mer lengths are used for de novo transcriptome assembly. We demonstrate its good performance by assembling de novo a published next-generation transcriptome sequence data set of Aedes aegypti, using the existing genome to check the accuracy of our method. The second method relies on the use of a reference proteome to improve the de novo assembly. We developed the Scaffolding using Translation Mapping (STM) method that uses mapping against the closest available reference proteome for scaffolding contigs that map onto the same protein. In a controlled experiment using simulated data, we show that the STM method considerably improves the assembly, with few errors. We applied these two methods to assemble the transcriptome of the non-model catfish Loricaria gr. cataphracta. Using the Multiple-k and STM methods, the assembly increases in contiguity and in gene identification, showing that our methods clearly improve quality and can be widely used. The new methods were used to assemble successfully the transcripts of the core set of genes regulating tooth development in vertebrates, while classic de novo assembly failed.

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CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors.

Publication Date: 2010 Aug 6 PMID: 20693478
Authors: Corbo, J. C. - Lawrence, K. A. - Karlstetter, M. - Myers, C. A. - Abdelaziz, M. - Dirkes, W. - Weigelt, K. - Seifert, M. - Benes, V. - Fritsche, L. G. - Weber, B. H. - Langmann, T.
Journal: Genome Res

Approximately 98% of mammalian DNA is non-coding, yet we understand relatively little about the function of this enigmatic portion of the genome. The cis-regulatory elements that control gene expression reside in non-coding regions and can be identified by mapping the binding sites of tissue-specific transcription factors. Cone-rod homeobox (CRX) is a key transcription factor in photoreceptor differentiation and survival, but its in vivo targets are largely unknown. Here we used ChIP-Seq on CRX to identify thousands of cis-regulatory regions around photoreceptor genes in adult mouse retina. CRX directly regulates downstream photoreceptor transcription factors and their target genes via a network of spatially distributed regulatory elements around each locus. CRX-bound regions act in a synergistic fashion to activate transcription and contain multiple CRX binding sites which interact in a spacing- and orientation-dependent manner to fine-tune transcript levels. CRX ChIP-Seq was also performed on Nrl(-/-) retinas which represent an enriched source of cone photoreceptors. Comparison with the wild-type ChIP-Seq dataset identified numerous rod- and cone-specific CRX-bound regions as well as many shared elements. Thus, CRX combinatorially orchestrates the transcriptional networks of both rods and cones by coordinating the expression of photoreceptor genes including most retinal disease genes. In addition, this study pinpoints thousands of non-coding regions of relevance to both Mendelian and complex retinal disease.

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Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2.

Publication Date: 2010 Sep PMID: 20688780
Authors: Nissenbaum, J. - Devor, M. - Seltzer, Z. - Gebauer, M. - Michaelis, M. - Tal, M. - Dorfman, R. - Abitbul-Yarkoni, M. - Lu, Y. - Elahipanah, T. - Delcanho, S. - Minert, A. - Fried, K. - Persson, A. K. - Shpigler, H. - Shabo, E. - Yakir, B. - Pisante, A. - Darvasi, A.
Journal: Genome Res

Chronic neuropathic pain is affected by specifics of the precipitating neural pathology, psychosocial factors, and by genetic predisposition. Little is known about the identity of predisposing genes. Using an integrative approach, we discovered that CACNG2 significantly affects susceptibility to chronic pain following nerve injury. CACNG2 encodes for stargazin, a protein intimately involved in the trafficking of glutamatergic AMPA receptors. The protein might also be a Ca(2+) channel subunit. CACNG2 has previously been implicated in epilepsy. Initially, using two fine-mapping strategies in a mouse model (recombinant progeny testing [RPT] and recombinant inbred segregation test [RIST]), we mapped a pain-related quantitative trait locus (QTL) (Pain1) into a 4.2-Mb interval on chromosome 15. This interval includes 155 genes. Subsequently, bioinformatics and whole-genome microarray expression analysis were used to narrow the list of candidates and ultimately to pinpoint Cacng2 as a likely candidate. Analysis of stargazer mice, a Cacng2 hypomorphic mutant, provided electrophysiological and behavioral evidence for the gene's functional role in pain processing. Finally, we showed that human CACNG2 polymorphisms are associated with chronic pain in a cohort of cancer patients who underwent breast surgery. Our findings provide novel information on the genetic basis of neuropathic pain and new insights into pain physiology that may ultimately enable better treatments.

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Genome-wide mapping of nuclear mitochondrial DNA sequences links DNA replication origins to chromosomal double-strand break formation in Schizosaccharomyces pombe.

Publication Date: 2010 Sep PMID: 20688779
Authors: Lenglez, S. - Hermand, D. - Decottignies, A.
Journal: Genome Res

Chromosomal double-strand breaks (DSBs) threaten genome integrity and repair of these lesions is often mutagenic. How and where DSBs are formed is a major question conveniently addressed in simple model organisms like yeast. NUMTs, nuclear DNA sequences of mitochondrial origin, are present in most eukaryotic genomes and probably result from the capture of mitochondrial DNA (mtDNA) fragments into chromosomal breaks. NUMT formation is ongoing and was reported to cause de novo human genetic diseases. Study of NUMTs is likely to contribute to the understanding of naturally occurring chromosomal breaks. We show that Schizosaccharomyces pombe NUMTs are exclusively located in noncoding regions with no preference for gene promoters and, when located into promoters, do not affect gene transcription level. Strikingly, most noncoding regions comprising NUMTs are also associated with a DNA replication origin (ORI). Chromatin immunoprecipitation experiments revealed that chromosomal NUMTs are probably not acting as ORI on their own but that mtDNA insertions occurred directly next to ORIs, suggesting that these loci may be prone to DSB formation. Accordingly, induction of excessive DNA replication origin firing, a phenomenon often associated with human tumor formation, resulted in frequent nucleotide deletion events within ORI3001 subtelomeric chromosomal locus, illustrating a novel aspect of DNA replication-driven genomic instability. How mtDNA is fragmented is another important issue that we addressed by sequencing experimentally induced NUMTs. This highlighted regions of S. pombe mtDNA prone to breaking. Together with an analysis of human NUMTs, we propose that these fragile sites in mtDNA may correspond to replication pause sites.

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Genomic signatures of germline gene expression.

Publication Date: 2010 Aug 4 PMID: 20686123
Authors: McVicker, G. - Green, P.
Journal: Genome Res

Transcribed regions in the human genome differ from adjacent intergenic regions in transposable element density, crossover rates, and asymmetric substitution and sequence composition patterns. We tested whether these differences reflect selection or are instead a byproduct of germline transcription using publicly available gene expression data from a variety of germline and somatic tissues. Crossover rate shows a strong negative correlation with gene expression in meiotic tissues, suggesting that crossover is inhibited by transcription. Strand biased composition (G+T content) and A-->G vs. T-->C substitution asymmetry are both positively correlated with germline gene expression. We find no evidence for a strand bias in allele frequency data, implying that the substitution asymmetry reflects a mutation rather than a fixation bias. The density of transposable elements is positively correlated with germline expression, suggesting that such elements preferentially insert into regions that are actively transcribed. For each of the features examined our analyses favor a non-selective explanation for the observed trends, and point to the role of germline gene expression in shaping the mammalian genome.

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Origins, evolution, and phenotypic impact of new genes.

Publication Date: 2010 Aug 13 PMID: 20651121
Authors: Kaessmann, H.
Journal: Genome Res

Ever since the pre-molecular era, the birth of new genes with novel functions has been considered to be a major contributor to adaptive evolutionary innovation. Here, I review the origin and evolution of new genes and their functions in eukaryotes, an area of research that has made rapid progress in the past decade thanks to the genomics revolution. Indeed, recent work has provided initial whole-genome views of the different types of new genes for a large number of different organisms. The array of mechanisms underlying the origin of new genes is compelling, extending way beyond the traditionally well-studied source of gene duplication. Thus, it was shown that novel genes also regularly arose from messenger RNAs of ancestral genes, protein-coding genes metamorphosed into new RNA genes, genomic parasites were co-opted as new genes, and that both protein and RNA genes were composed from scratch (i.e., from previously nonfunctional sequences). These mechanisms then also contributed to the formation of numerous novel chimeric gene structures. Detailed functional investigations uncovered different evolutionary pathways that led to the emergence of novel functions from these newly minted sequences and, with respect to animals, attributed a potentially important role to one specific tissue-the testis-in the process of gene birth. Remarkably, these studies also demonstrated that novel genes of the various types significantly impacted the evolution of cellular, physiological, morphological, behavioral, and reproductive phenotypic traits. Consequently, it is now firmly established that new genes have indeed been major contributors to the origin of adaptive evolutionary novelties.

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The ANISEED database: Digital representation, formalization, and elucidation of a chordate developmental program.

Publication Date: 2010 Aug 13 PMID: 20647237
Authors: Tassy, O. - Dauga, D. - Daian, F. - Sobral, D. - Robin, F. - Khoueiry, P. - Salgado, D. - Fox, V. - Caillol, D. - Schiappa, R. - Laporte, B. - Rios, A. - Luxardi, G. - Kusakabe, T. - Joly, J. S. - Darras, S. - Christiaen, L. - Contensin, M. - Auger, H. - Lamy, C. - Hudson, C. - Rothbacher, U. - Gilchrist, M. J. - Makabe, K. W. - Hotta, K. - Fujiwara, S. - Satoh, N. - Satou, Y. - Lemaire, P.
Journal: Genome Res

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

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The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data.

Publication Date: 2010 Sep PMID: 20644199
Authors: McKenna, A. - Hanna, M. - Banks, E. - Sivachenko, A. - Cibulskis, K. - Kernytsky, A. - Garimella, K. - Altshuler, D. - Gabriel, S. - Daly, M. - Depristo, M. A.
Journal: Genome Res

Next-generation DNA sequencing (NGS) projects, such as the 1000 Genomes Project, are already revolutionizing our understanding of genetic variation among individuals. However, the massive data sets generated by NGS-the 1000 Genome pilot alone includes nearly five terabases-make writing feature-rich, efficient, and robust analysis tools difficult for even computationally sophisticated individuals. Indeed, many professionals are limited in the scope and the ease with which they can answer scientific questions by the complexity of accessing and manipulating the data produced by these machines. Here, we discuss our Genome Analysis Toolkit (GATK), a structured programming framework designed to ease the development of efficient and robust analysis tools for next-generation DNA sequencers using the functional programming philosophy of MapReduce. The GATK provides a small but rich set of data access patterns that encompass the majority of analysis tool needs. Separating specific analysis calculations from common data management infrastructure enables us to optimize the GATK framework for correctness, stability, and CPU and memory efficiency and to enable distributed and shared memory parallelization. We highlight the capabilities of the GATK by describing the implementation and application of robust, scale-tolerant tools like coverage calculators and single nucleotide polymorphism (SNP) calling. We conclude that the GATK programming framework enables developers and analysts to quickly and easily write efficient and robust NGS tools, many of which have already been incorporated into large-scale sequencing projects like the 1000 Genomes Project and The Cancer Genome Atlas.

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Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples.

Publication Date: 2010 Sep PMID: 20627893
Authors: Varley, K. E. - Mitra, R. D.
Journal: Genome Res

Aberrant DNA methylation frequently occurs at gene promoters during cancer progression. It is important to identify these loci because they are often misregulated and drive tumorigenesis. Bisulfite sequencing is the most direct and highest resolution assay for identifying aberrant promoter methylation. Recently, genomic capture methods have been combined with next-generation sequencing to enable genome-scale surveys of methylation in individual samples. However, it is challenging to validate candidate loci identified by these approaches because an efficient method to bisulfite sequence more than 50 differentially methylated loci across a large number of samples does not exist. To address this problem, we developed Bisulfite Patch PCR, which enables highly multiplexed bisulfite PCR and sequencing across many samples. Using this method, we successfully amplified 100% of 94 targeted gene promoters simultaneously in the same reaction. By incorporating sample-specific DNA barcodes into the amplicons, we analyzed 48 samples in a single run of the 454 Life Sciences (Roche) FLX sequencer. The method requires small amounts of starting DNA (250 ng) and does not require a shotgun library construction. The method was highly specific; 90% of sequencing reads aligned to targeted loci. The targeted promoters were from genes that are frequently mutated in breast and colon cancer, and the samples included breast and colon tumor and adjacent normal tissue. This approach allowed us to identify nine gene promoters that exhibit tumor-specific DNA methylation defects that occur frequently in colon and breast cancer. We also analyzed single nucleotide polymorphisms to observe DNA methylation that accumulated on specific alleles during tumor development. This method is broadly applicable for studying DNA methylation across large numbers of patient samples using next-generation sequencing.

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Function annotation of the rice transcriptome at single-nucleotide resolution by RNA-seq.

Publication Date: 2010 Sep PMID: 20627892
Authors: Lu, T. - Lu, G. - Fan, D. - Zhu, C. - Li, W. - Zhao, Q. - Feng, Q. - Zhao, Y. - Guo, Y. - Li, W. - Huang, X. - Han, B.
Journal: Genome Res

The functional complexity of the rice transcriptome is not yet fully elucidated, despite many studies having reported the use of DNA microarrays. Next-generation DNA sequencing technologies provide a powerful approach for mapping and quantifying the transcriptome, termed RNA sequencing (RNA-seq). In this study, we applied RNA-seq to globally sample transcripts of the cultivated rice Oryza sativa indica and japonica subspecies for resolving the whole-genome transcription profiles. We identified 15,708 novel transcriptional active regions (nTARs), of which 51.7% have no homolog to public protein data and >63% are putative single-exon transcripts, which are highly different from protein-coding genes (<20%). We found that approximately 48% of rice genes show alternative splicing patterns, a percentage considerably higher than previous estimations. On the basis of the available rice gene models, 83.1% (46,472 genes) of the current rice gene models were validated by RNA-seq, and 6228 genes were identified to be extended at the 5' and/or 3' ends by at least 50 bp. Comparative transcriptome analysis demonstrated that 3464 genes exhibited differential expression patterns. The ratio of SNPs with nonsynonymous/synonymous mutations was nearly 1:1.06. In total, we interrogated and compared transcriptomes of the two rice subspecies to reveal the overall transcriptional landscape at maximal resolution.

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TATA is a modular component of synthetic promoters.

Publication Date: 2010 Aug 23 PMID: 20627890
Authors: Mogno, I. - Vallania, F. - Mitra, R. D. - Cohen, B. A.
Journal: Genome Res

The expression of most genes is regulated by multiple transcription factors. The interactions between transcription factors produce complex patterns of gene expression that are not always obvious from the arrangement of cis-regulatory elements in a promoter. One critical element of promoters is the TATA box, the docking site for the RNA polymerase holoenzyme. Using a synthetic promoter system coupled to a thermodynamic model of combinatorial regulation, we analyze the effects of different strength TATA boxes on various aspects of combinatorial cis-regulation. The thermodynamic model explains 75% of the variance in gene expression in synthetic promoter libraries with different strength TATA boxes, suggesting that many of the salient aspects of cis-regulation are captured by the model. Our results demonstrate that the effect of changing the TATA box on gene expression is the same for all synthetic promoters regardless of the arrangement of cis-regulatory sites we studied. Our analysis also showed that in our synthetic system the strength of the RNA polymerase-TATA interaction does not alter the combinatorial interactions between transcription factors, or between transcription factors and RNA polymerase. Finally, we show that although stronger TATA boxes increase expression in a predictable fashion, stronger TATA boxes have very little effect on noise in our synthetic promoters, regardless of the arrangement of cis-regulatory sites. Our results support a modular model of promoter function, where cis-regulatory elements can be mixed and matched (programmed) with outcomes on expression that are predictable based on the rules of simple protein-protein and protein-DNA interactions.

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