Genome Research

Translating cancer 'omics' to improved outcomes.

Publication Date: 2012 Feb PMID: 22301133
Authors: Vucic, E. A. - Thu, K. L. - Robison, K. - Rybaczyk, L. A. - Chari, R. - Alvarez, C. E. - Lam, W. L.
Journal: Genome Res

The genomics era has yielded great advances in the understanding of cancer biology. At the same time, the immense complexity of the cancer genome has been revealed, as well as a striking heterogeneity at the whole-genome (or omics) level that exists between even histologically similar tumors. The vast accrual and public availability of multi-omics databases with associated clinical annotation including tumor histology, patient response, and outcome are a rich resource that has the potential to lead to rapid translation of high-throughput omics to improved overall survival. We focus on the unique advantages of a multidimensional approach to genomic analysis in this new high-throughput omics age and discuss the implications of the changing cancer demographic to translational omics research.

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Translational genomics: The challenge of developing cancer biomarkers.

Publication Date: 2012 Feb PMID: 22301132
Authors: Brooks, J. D.
Journal: Genome Res

Early detection and definitive treatment of cancer have been shown to decrease death and suffering in epidemiologic and intervention studies. Application of genomic approaches to many malignancies has produced thousands of candidate biomarkers for detection and prognostication, yet very few have become established in clinical practice. Fundamental issues related to tumor heterogeneity, cancer progression, natural history, and biomarker performance have provided challenges to biomarker development. Technical issues in biomarker assay detection limits, specificity, clinical deployment, and regulation have also slowed progress. The recent emergence of biomarkers and molecular imaging strategies for treatment selection and monitoring demonstrates the promise of cancer biomarkers. Organized efforts by interdisciplinary teams will spur progress in cancer diagnostics.

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An emerging toolkit for targeted cancer therapies.

Publication Date: 2012 Feb PMID: 22301131
Authors: Mills, G. B.
Journal: Genome Res

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Effects of sequence variation on differential allelic transcription factor occupancy and gene expression.

Publication Date: 2012 Feb 2 PMID: 22300769
Authors: Reddy, T. E. - Gertz, J. - Pauli, F. - Kucera, K. S. - Varley, K. E. - Newberry, K. M. - Marinov, G. K. - Mortazavi, A. - Williams, B. A. - Song, L. - Crawford, G. E. - Wold, B. J. - Willard, H. F. - Myers, R. M.
Journal: Genome Res

A complex interplay between transcription factors (TFs) and the genome is essential for proper regulation of transcription. However, directly connecting variation in genomic DNA sequence with variation in TF binding and gene expression is challenging due to limited knowledge of where TFs bind and environmental differences between individuals and cell types. To address this problem, we measured genome-wide differential allelic occupancy of 24 sequence-specific TFs and EP300 in a human lymphoblastoid cell line (LCL), GM12878. Overall, 5% of human TF binding sites have an allelic imbalance in occupancy. At many sites, TFs were clustered together on the genome in TF-binding hubs, where they occupied the same homolog in regions of especially open chromatin. While genetic variation in core TF binding motifs generally resulted in large allelic differences in TF occupancy, the majority of allelic differences in occupancy were subtle and associated with disruption of weak or non-canonical DNA binding motifs. We also measured genome-wide differential allelic expression of genes both with and without heterozygous exonic variants in the same cells. We found that genes with differential allelic expression were overall less expressed, not just in GM12878 cells, but also in a panel of unrelated human cell lines. Comparing TF occupancy with expression, we found a strong association between allelic occupancy and expression within 100 bp of transcription start sites (TSSs), and a weak association up to 100 kb away from TSSs. When compared to disease-associated regions identified in genome-wide association studies, sites of differential allelic occupancy were significantly enriched for variants associated with disease, particularly autoimmune disease. These results highlight the potential for allelic differences in TF occupancy to give functional insights into intergenic variants associated with disease. Our results have the potential to increase the power and interpretability of association studies by targeting functional intergenic variants in addition to protein coding sequences.

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Copy number variation of individual cattle genomes using next-generation sequencing.

Publication Date: 2012 Feb 2 PMID: 22300768
Authors: Bickhart, D. M. - Hou, Y. - Schroeder, S. G. - Alkan, C. - Cardone, M. F. - Matukumalli, L. K. - Song, J. - Schnabel, R. D. - Ventura, M. - Taylor, J. F. - Garcia, J. F. - Van Tassell, C. P. - Sonstegard, T. S. - Eichler, E. E. - Liu, G. E.
Journal: Genome Res

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one Holstein and one Hereford) and one indicine (Nelore) cattle. Within mapped chromosomal sequence, we identified 1,265 CNV regions comprising ~55.6 Mbp sequence - 476 of which (~38%) have not previously been reported. We validated this sequence-based CNV call set with aCGH, qPCR and FISH, achieving a validation rate of 82% and a false positive rate of 8%. We further estimated absolute copy numbers for genomic segments and annotated genes in each individual. Surveys of the top 25 most variable genes revealed that the Nelore individual had the lowest copy numbers in 13 cases (~52%, chi squared test; p value <0.05). In contrast, genes related to pathogen- and parasite-resistance, such as CATHL4 and ULBP17, were highly duplicated in the Nelore individual relative to the taurine cattle, while genes involved in lipid transport and metabolism, including APOL3 and FABP2, were highly duplicated in the beef breeds. These CNV regions also harbor genes like BPIFA2A (BSP30A) and WC1, suggesting that some CNVs may be associated with breed-specific differences in adaptation, health, and production traits. By providing the first individualized cattle CNV and segmental duplication maps and genome-wide gene copy number estimates, we enable future CNV studies into highly duplicated regions in the cattle genome.

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Extension of cortical synaptic development distinguishes humans from chimpanzees and macaques.

Publication Date: 2012 Feb 2 PMID: 22300767
Authors: Liu, X. - Somel, M. - Tang, L. - Yan, Z. - Jiang, X. - Guo, S. - Yuan, Y. - He, L. - Oleksiak, A. - Zhang, Y. - Li, N. - Hu, Y. - Chen, W. - Qiu, Z. - Paabo, S. - Khaitovich, P.
Journal: Genome Res

Over the course of ontogenesis, the human brain and human cognitive abilities develop in parallel, resulting in a phenotype strikingly distinct from that of other primates. Here, we used microarrays and RNA-sequencing to examine human-specific gene expression changes taking place during postnatal brain development in the prefrontal cortex and cerebellum of humans, chimpanzees, and rhesus macaques. We show that the most prominent human-specific expression change affects genes associated with synaptic functions and represents an extreme shift in the timing of synaptic development in the prefrontal cortex, but not the cerebellum. Consequently, peak expression of synaptic genes in the prefrontal cortex is shifted from <1 yr in chimpanzees and macaques to 5 yr in humans. This result was supported by protein expression profiles of synaptic density markers and by direct observation of synaptic density by electron microscopy. Mechanistically, the human-specific change in timing of synaptic development involves the MEF2A-mediated activity-dependent regulatory pathway. Evolutionarily, this change may have taken place after the split of the human and the Neanderthal lineages.

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VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome sequencing.

Publication Date: 2012 Feb 2 PMID: 22300766
Authors: Koboldt, D. C. - Zhang, Q. - Larson, D. E. - Shen, D. - McLellan, M. D. - Lin, L. - Miller, C. A. - Mardis, E. R. - Ding, L. - Wilson, R. K.
Journal: Genome Res

Cancer is a disease driven by genetic variation and mutation. Exome sequencing can be utilized for discovering these variants and mutations across hundreds of tumors. Here we present an analysis tool, VarScan 2, for the detection of somatic mutations and copy number alterations (CNAs) in exome data from tumor-normal pairs. Unlike most current approaches, our algorithm reads data from both samples simultaneously; a heuristic and statistical algorithm detects sequence variants and classifies them by somatic status (germline, somatic, or LOH); while a comparison of normalized read depth delineates relative copy number changes. We apply these methods to the analysis of exome sequence data from 151 high-grade ovarian tumors characterized as part of the Cancer Genome Atlas (TCGA). We validated some 7790 somatic coding mutations, achieving 93% sensitivity and 85% precision for single nucleotide variant (SNV) detection. Exome-based CNA analysis identified 29 large-scale alterations and 619 focal events per tumor on average. As in our previous analysis of these data, we observed frequent amplification of oncogenes (e.g., CCNE1, MYC) and deletion of tumor suppressors (NF1, PTEN, and CDKN2A). We searched for additional recurrent focal CNAs using the correlation matrix diagonal segmentation (CMDS) algorithm, which identified 424 significant events affecting 582 genes. Taken together, our results demonstrate the robust performance of VarScan 2 for somatic mutation and CNA detection and shed new light on the landscape of genetic alterations in ovarian cancer.

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A vast collection of microbial genes that are toxic to bacteria.

Publication Date: 2012 Feb 1 PMID: 22300632
Authors: Kimelman, A. - Levy, A. - Sberro, H. - Kidron, S. - Leavitt, A. - Amitai, G. - Yoder-Himes, D. - Wurtzel, O. - Zhu, Y. - Rubin, E. - Sorek, R.
Journal: Genome Res

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes our assays revealed novel toxins and restriction enzymes, and new classes of small non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator dnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.

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Age-associated DNA methylation in pediatric populations.

Publication Date: 2012 Feb 1 PMID: 22300631
Authors: Alisch, R. S. - Barwick, B. G. - Chopra, P. - Myrick, L. K. - Satten, G. A. - Conneely, K. N. - Warren, S. T.
Journal: Genome Res

DNA methylation (DNAm) plays diverse roles in human biology, but this dynamic epigenetic mark remains far from fully characterized. Although earlier studies uncovered loci that undergo age-associated DNAm changes in adults, little is known about such changes during childhood. Despite profound DNAm plasticity during embryogenesis, monozygotic twins show indistinguishable childhood methylation, suggesting DNAm is highly coordinated throughout early development. Here we examine the methylation of 27,578 CpG dinucleotides in peripheral blood DNA from a cross-sectional study of 398 boys, aged 3 to 17 years, and find significant age-associated changes in DNAm at 2,078 loci. These findings correspond well with pyrosequencing data and replicate in a second pediatric population (N=78). Moreover, we report a deficit of age-related loci on the X chromosome, a preference for specific nucleotides immediately surrounding the interrogated CpG dinucleotide, and a primary association with developmental and immune ontological functions. Meta-analysis (N=1,158) with two adult populations reveals that despite a significant overlap of age-associated loci, most methylation changes do not follow a lifelong linear pattern due to a three- to four-fold higher rate of change in children compared to adults; consequently the vast majority of changes are more accurately modeled as a function of logarithmic age. We therefore conclude that age-related DNAm changes in peripheral blood occur more rapidly during childhood and are imperfectly accounted for by statistical corrections that are linear in age, further suggesting that future DNAm studies should be matched closely for age.

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Microevolution of extensively drug-resistant tuberculosis in Russia.

Publication Date: 2012 Jan 31 PMID: 22294518
Authors: Casali, N. - Nikolayevskyy, V. - Balabanova, Y. - Ignatyeva, O. - Kontsevaya, I. - Harris, S. R. - Bentley, S. D. - Parkhill, J. - Nejentsev, S. - Hoffner, S. E. - Horstmann, R. D. - Brown, T. - Drobniewski, F.
Journal: Genome Res

Extensively drug resistant tuberculosis (XDR TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2,348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia revealed that 72% belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that evolution of this population is primarily driven by drug therapy. Resistance genotypes showed strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted.

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A multiplicity of factors contributes to selective RNA polymerase III occupancy of a subset of RNA polymerase III genes in mouse liver.

Publication Date: 2012 Jan 27 PMID: 22287103
Authors: Canella, D. - Bernasconi, D. - Gilardi, F. - Lemartelot, G. - Migliavacca, E. - Praz, V. - Cousin, P. - Delorenzi, M. - Hernandez, N.
Journal: Genome Res

The genomic loci occupied by RNA polymerase (RNAP) III have been characterized in human culture cells by genome-wide chromatin immunoprecipitations, followed by deep sequencing (ChIP-seq). These studies have shown that only approximately 40% of the annotated 622 human tRNA genes and pseudogenes are occupied by RNAP-III, and that these genes are often in open chromatin regions rich in active RNAP-II transcription units. We have used ChIP-seq to characterize RNAP-III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver RNAP-III-occupied loci including a conserved mammalian interspersed repeat (MIR) as a potential regulator of an RNAP-III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse RNAP-III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence, can strongly affect RNAP-III occupancy of tRNA genes. They reveal correlations with various genomic features that explain the observed variation of 81% of tRNA scores. In mouse liver, loci represented in the NCBI37/mm9 genome assembly that are clearly occupied by RNAP-III comprise 50 Rn5s (5S RNA) genes, 14 known non-tRNA RNAP-III genes, nine Rn4.5s (4.5S RNA) genes, and 29 SINEs. Moreover, out of the 433 annotated tRNA genes, half are occupied by RNAP-III. Transfer RNA gene expression levels reflect both an underlying genomic organization conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes.

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Retrotransposon profiling of RNA polymerase III initiation sites.

Publication Date: 2012 Jan 27 PMID: 22287102
Authors: Qi, X. - Daily, K. - Nguyen, K. - Wang, H. - Mayhew, D. - Rigor, P. - Forouzan, S. - Johnston, M. - Mitra, R. D. - Baldi, P. - Sandmeyer, S.
Journal: Genome Res

Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. In order to determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes.

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Evolutionary rate covariation reveals shared functionality and co-expression of genes.

Publication Date: 2012 Jan 27 PMID: 22287101
Authors: Clark, N. L. - Alani, E. - Aquadro, C. F.
Journal: Genome Res

Evolutionary rate covariation (ERC) is a phylogenetic signature that reflects the covariation of a pair of proteins over evolutionary time. ERC is typically elevated between interacting proteins, and so is a promising signature to characterize molecular and functional interactions across the genome. ERC is often assumed to result from compensatory changes at interaction interfaces (i.e. intermolecular coevolution); however, its origin is still unclear and is likely to be complex. Here, we determine the biological factors responsible for ERC in a proteome-wide dataset of 4,459 proteins in 18 budding yeast species. We show that direct physical interaction is not required to produce ERC, because we observe strong correlations between non-interacting but co-functional enzymes. We also demonstrate that ERC is uniformly distributed along the protein primary sequence, suggesting that intermolecular coevolution is not generally responsible for ERC between physically interacting proteins. Using multivariate analysis, we show that a pair of proteins is likely to exhibit ERC if they share a biological function or if their expression levels coevolve between species. Thus, ERC indicates shared function and co-expression of protein pairs and not necessarily coevolution between sites as has been assumed in previous studies. This full interpretation of ERC now provides us with a powerful tool to assign uncharacterized proteins to functional groups and to determine the interconnectedness between entire genetic pathways.

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Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs.

Publication Date: 2012 Jan 26 PMID: 22282571
Authors: Sharifpoor, S. - van Dyk, D. - Costanzo, M. - Baryshnikova, A. - Friesen, H. - Douglas, A. C. - Youn, J. Y. - Vandersluis, B. - Myers, C. L. - Papp, B. - Boone, C. - Andrews, B. J.
Journal: Genome Res

A combinatorial genetic perturbation strategy was applied to interrogate the yeast kinome on a genome-wide scale. We assessed the global effects of gene overexpression or gene deletion to map an integrated genetic interaction network of Synthetic Dosage Lethal (SDL) and loss-of-function genetic interactions (GIs) for 92 kinases, producing a meta-network of 8,700 GIs enriched for pathways known to be regulated by cognate kinases. Kinases most sensitive to dosage perturbations had constitutive cell cycle or cell polarity functions under standard growth conditions. Condition-specific screens confirmed that the spectrum of kinase dosage interactions can be expanded substantially in activating conditions. An integrated network composed of systematic SDL, negative and positive loss-of-function GIs and literature curated kinase-substrate interactions revealed kinase-dependent regulatory motifs predictive of novel gene-specific phenotypes. Our study provides a valuable resource to unravel novel functional relationships and pathways regulated by kinases and outlines a general strategy for deciphering mutant phenotypes from large-scale genetic interaction networks.

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Unified modeling of gene duplication, loss, and coalescence using a locus tree.

Publication Date: 2012 Jan 23 PMID: 22271778
Authors: Rasmussen, M. D. - Kellis, M.
Journal: Genome Res

Gene phylogenies provide a rich source of information about the way evolution shapes genomes, populations, and phenotypes. In addition to substitutions, evolutionary events such as gene duplication and loss (as well as horizontal transfer) play a major role in gene evolution, and many phylogenetic models have been developed in order to reconstruct and study these events. However, these models typically make the simplifying assumption that population-related effects such as incomplete lineage sorting (ILS) are negligible. While this assumption may have been reasonable in some settings, it has become increasingly problematic as increased genome sequencing has led to denser phylogenies, where effects such as ILS are more prominent. To address this challenge, we present a new probabilistic model, DLCoal, that defines gene duplication and loss in a population setting, such that coalescence and ILS can be directly addressed. Interestingly, this model implies that in addition to the usual gene tree and species tree there exists a third tree, the locus tree, which will likely have many applications. Using this model, we develop the first general reconciliation method that accurately infers gene duplications and losses in the presence of ILS, and we show its improved inference of orthologs, paralogs, duplications, and losses for a variety of clades, including flies, fungi, and primates. Also, our simulations show that gene duplications increase the frequency of ILS, further illustrating the importance of a joint model. Going forward, we believe this unified model can offer insights to questions in both phylogenetics and population genetics.

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The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients.

Publication Date: 2012 Jan 20 PMID: 22267523
Authors: Jiang, Z. - Jhunjhunwala, S. - Liu, J. - Haverty, P. M. - Kennemer, M. I. - Guan, Y. - Lee, W. - Carnevali, P. - Stinson, J. - Johnson, S. - Diao, J. - Yeung, S. - Jubb, A. - Ye, W. - Wu, T. D. - Kapadia, S. B. - de Sauvage, F. J. - Gentleman, R. C. - Stern, H. M. - Seshagiri, S. - Pant, K. P. - Modrusan, Z. - Ballinger, D. G. - Zhang, Z.
Journal: Genome Res

Hepatitis B virus (HBV) infection is a leading risk factor for hepatocellular carcinoma (HCC). HBV integration into the host genome has been reported but its scale, impact and contribution to HCC development is not clear. Here, we sequenced the tumor and non-tumor genomes (>80X coverage) and transcriptomes of four HCC patients and identified 255 HBV integration sites. Increased sequencing to 240X coverage revealed a proportionally higher number of integration sites. Clonal expansion of HBV-integrated hepatocytes was found specifically in tumor samples. We observe a diverse collection of genomic perturbations near viral integration sites, including direct gene disruption, viral promoter-driven human transcription, viral-human transcript fusion and DNA copy number alteration. Thus, we report the most comprehensive characterization of HBV integration in hepatocellular carcinoma patients. Such widespread random viral integration will likely increase carcinogenic opportunities in HBV-infected individuals.

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Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture.

Publication Date: 2012 Jan 20 PMID: 22267522
Authors: Rohland, N. - Reich, D.
Journal: Genome Res

Improvements in technology have reduced the cost of DNA sequencing to the point that the limiting factor for many experiments is the time and reagent cost of sample preparation. We present an approach in which 192 sequencing libraries can be produced in a single day of technician time at cost of about $15 per sample. These libraries are effective not only for low-pass whole genome sequencing, but also for simultaneously enriching them in pools of approximately hundred individually barcoded samples for a subset of the genome without substantial loss in efficiency of target capture. We illustrate the power and effectiveness of this approach on about 2,000 samples from a prostate cancer study.

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The TAGteam motif facilitates binding of 21 sequence-specific transcription factors in the Drosophila embryo.

Publication Date: 2012 Jan 13 PMID: 22247430
Authors: Satija, R. - Bradley, R. K.
Journal: Genome Res

Highly overlapping patterns of genome-wide binding of many distinct transcription factors have been observed in worms, insects, and mammals, but the origins and consequences of this overlapping binding remain unclear. Analyzing chromatin immunoprecipitation datasets from 21 sequence-specific transcription factors active in the Drosophila embryo, we found that binding of all factors exhibits a dose-dependent relationship with "TAGteam" sequence motifs bound by the zinc-finger protein Vielfaltig, also known as Zelda, a recently discovered activator of the zygotic genome. TAGteam motifs are present and well-conserved in highly bound regions, and are associated with transcription factor binding even in the absence of canonical recognition motifs for these factors. Furthermore, levels of binding in promoters and enhancers of zygotically transcribed genes are directly proportional to RNA polymerase II occupancy and gene expression levels. Our results suggest that Vielfaltig acts as a master regulator of early development by facilitating the establishment of genome-wide patterns of transcription factor binding, which drive global gene expression.

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HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter.

Publication Date: 2012 Jan 26 PMID: 22234890
Authors: Visser, M. - Kayser, M. - Palstra, R. J.
Journal: Genome Res

Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases.

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An ancient genomic regulatory block conserved across bilaterians and its dismantling in tetrapods by retrogene replacement.

Publication Date: 2012 Jan 10 PMID: 22234889
Authors: Maeso, I. - Irimia, M. - Tena, J. J. - Gonzalez-Perez, E. - Tran, D. - Ravi, V. - Venkatesh, B. - Campuzano, S. - Gomez-Skarmeta, J. L. - Garcia-Fernandez, J.
Journal: Genome Res

Developmental genes are regulated by complex, distantly located, cis-regulatory modules (CRMs), often forming genomic regulatory blocks (GRBs) that are conserved among vertebrates and among insects. We have investigated GRBs associated with Iroquois homeobox genes in 39 metazoans. Despite 600 million years of independent evolution, Iroquois genes are linked to ankyrin-repeat containing Sowah genes in nearly all studied bilaterians. We show that Iroquois-specific CRMs populate the Sowah locus, suggesting that regulatory constraints underlie the maintenance of the Iroquois-Sowah syntenic block. Surprisingly, tetrapod Sowah orthologs are intronless and not associated with Iroquois, however, teleost and elephant shark data demonstrate that this is a derived feature and that many Iroquois-CRMs were ancestrally located within Sowah introns. Retroposition, gene and genome duplication have allowed selective elimination of Sowah exons from the Iroquois regulatory landscape while keeping associated CRMs, resulting in large associated gene deserts. These results highlight the importance of CRMs in imposing constraints to genome architecture, even across large phylogenetic distances, and of gene duplication-mediated genetic redundancy to disentangle these constraints, increasing genomic plasticity.

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A nucleosomal surface defines an integration hotspot for the Saccharomyces cerevisiae Ty1 retrotransposon.

Publication Date: 2012 Jan 31 PMID: 22219511
Authors: Baller, J. A. - Gao, J. - Stamenova, R. - Curcio, M. J. - Voytas, D. F.
Journal: Genome Res

Ty1, the most abundant retrotransposon in Saccharomyces cerevisiae, integrates preferentially upstream of genes transcribed by RNA polymerase III (Pol III). Targeting is likely due to interactions between the Ty1 integration complex and a feature of chromatin characteristic of sites of Pol III transcription. To better understand Ty1 targeting determinants, >150,000 Ty1 insertions were mapped onto the S. cerevisiae genome sequence. Logistic regression was used to assess relationships between patterns of Ty1 integration and various genomic features, including genome-wide data sets of histone modifications and transcription-factor binding sites. Nucleosomes were positively associated with Ty1 insertions, and fine-scale mapping of insertions upstream of genes transcribed by Pol III indicated that Ty1 preferentially integrates into nucleosome-bound DNA near the H2A/H2B interface. Outside of genes transcribed by Pol III, Ty1 avoids coding sequences, a pattern that is not due to selection, but rather reflects a preference for nucleosome-rich sites flanking genes. Ty1 insertion sites were also mapped in four mutant lines that affect Ty1 transposition frequency or integration specificity (rrm3Delta, hos2Delta, rtt109Delta, and rad6Delta). Patterns of integration were largely preserved in the mutants, although significantly more insertions into coding sequences were observed in the rad6Delta strain, suggesting a loosening of target specificity in this mutant that lacks an enzyme involved in ubiquitinating H2A. Overall, our data suggest that nucleosomes are necessary for Ty1 integration, and that a secondary factor, likely a histone modification or nucleosome-bound factor enriched at sites of Pol III transcription, determines preferred target sites.

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Retrotransposon Ty1 integration targets specifically positioned asymmetric nucleosomal DNA segments in tRNA hotspots.

Publication Date: 2012 Jan 9 PMID: 22219510
Authors: Mularoni, L. - Zhou, Y. - Bowen, T. - Gangadharan, S. - Wheelan, S. - Boeke, J.
Journal: Genome Res

The Saccharomyces cerevisiae genome contains about 35 copies of dispersed retrotransposons called Ty1 elements. Ty1 elements target regions lying upstream of tRNA genes and other Pol III-transcribed genes when retrotransposing to new sites. We used deep sequencing of Ty1-flanking sequence amplicons to characterize Ty1 integration. Surprisingly, some insertions were found in mitochondrial DNA sequences, presumably reflecting insertion into mitochondrial DNA segments that had migrated to the nucleus. The overwhelming majority of insertions were associated with the 5' regions of Pol III transcribed genes; alignment of Ty1 insertion sites revealed a strong sequence motif centered on but extending beyond the target site duplication. A strong sequence-independent preference for nucleosomal integration sites was observed, in distinction to the preferences of the Hermes DNA transposon engineered to jump in yeast and the Tf1 retrotransposon of Schizosaccharomyces pombe, both of which prefer nucleosome free regions. Remarkably, an exquisitely specific relationship between Ty1 integration and nucleosomal position was revealed by alignment of hotspot Ty1 insertion positions regions to peak nucleosome positions, geographically implicating nucleosomal DNA segments at specific positions on the nucleosome lateral surface as targets, near the bottom of the nucleosome. The specificity is observed in the three tRNA 5'-proximal nucleosomes, with insertion frequency dropping off sharply 5' to the tRNA gene. The sites are disposed asymmetrically on the nucleosome, ruling out several simple molecular models for Ty1 targeting, and instead suggesting association with a dynamic process such as nucleosome remodeling.

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Comparative RNA sequencing reveals substantial genetic variation in endangered primates.

Publication Date: 2012 Jan 31 PMID: 22207615
Authors: Perry, G. H. - Melsted, P. - Marioni, J. C. - Wang, Y. - Bainer, R. - Pickrell, J. K. - Michelini, K. - Zehr, S. - Yoder, A. D. - Stephens, M. - Pritchard, J. K. - Gilad, Y.
Journal: Genome Res

Comparative genomic studies in primates have yielded important insights into the evolutionary forces that shape genetic diversity and revealed the likely genetic basis for certain species-specific adaptations. To date, however, these studies have focused on only a small number of species. For the majority of nonhuman primates, including some of the most critically endangered, genome-level data are not yet available. In this study, we have taken the first steps toward addressing this gap by sequencing RNA from the livers of multiple individuals from each of 16 mammalian species, including humans and 11 nonhuman primates. Of the nonhuman primate species, five are lemurs and two are lorisoids, for which little or no genomic data were previously available. To analyze these data, we developed a method for de novo assembly and alignment of orthologous gene sequences across species. We assembled an average of 5721 gene sequences per species and characterized diversity and divergence of both gene sequences and gene expression levels. We identified patterns of variation that are consistent with the action of positive or directional selection, including an 18-fold enrichment of peroxisomal genes among genes whose regulation likely evolved under directional selection in the ancestral primate lineage. Importantly, we found no relationship between genetic diversity and endangered status, with the two most endangered species in our study, the black and white ruffed lemur and the Coquerel's sifaka, having the highest genetic diversity among all primates. Our observations imply that many endangered lemur populations still harbor considerable genetic variation. Timely efforts to conserve these species alongside their habitats have, therefore, strong potential to achieve long-term success.

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Ultraconserved elements are novel phylogenomic markers that resolve placental mammal phylogeny when combined with species-tree analysis.

Publication Date: 2012 Feb 2 PMID: 22207614
Authors: McCormack, J. E. - Faircloth, B. C. - Crawford, N. G. - Gowaty, P. A. - Brumfield, R. T. - Glenn, T. C.
Journal: Genome Res

Phylogenomics offers the potential to fully resolve the Tree of Life, but increasing genomic coverage also reveals conflicting evolutionary histories among genes, demanding new analytical strategies for elucidating a single history of life. Here, we outline a phylogenomic approach using a novel class of phylogenetic markers derived from ultraconserved elements and flanking DNA. Using species-tree analysis that accounts for discord among hundreds of independent loci, we show that this class of marker is useful for recovering deep-level phylogeny in placental mammals. In broad outline, our phylogeny agrees with recent phylogenomic studies of mammals, including several formerly controversial relationships. Our results also inform two outstanding questions in placental mammal phylogeny involving rapid speciation, where species-tree methods are particularly needed. Contrary to most phylogenomic studies, our study supports a first-diverging placental mammal lineage that includes elephants and tenrecs (Afrotheria). The level of conflict among gene histories is consistent with this basal divergence occurring in or near a phylogenetic "anomaly zone" where a failure to account for coalescent stochasticity will mislead phylogenetic inference. Addressing a long-standing phylogenetic mystery, we find some support from a high genomic coverage data set for a traditional placement of bats (Chiroptera) sister to a clade containing Perissodactyla, Cetartiodactyla, and Carnivora, and not nested within the latter clade, as has been suggested recently, although other results were conflicting. One of the most remarkable findings of our study is that ultraconserved elements and their flanking DNA are a rich source of phylogenetic information with strong potential for application across Amniotes.

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A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing.

Publication Date: 2012 Jan 23 PMID: 22194472
Authors: Ju, Y. S. - Lee, W. C. - Shin, J. Y. - Lee, S. - Bleazard, T. - Won, J. K. - Kim, Y. T. - Kim, J. I. - Kang, J. H. - Seo, J. S.
Journal: Genome Res

The identification of the molecular events that drive cancer transformation is essential to the development of targeted agents that improve the clinical outcome of lung cancer. Many studies have reported genomic driver mutations in non-small-cell lung cancers (NSCLCs) over the past decade; however, the molecular pathogenesis of >40% of NSCLCs is still unknown. To identify new molecular targets in NSCLCs, we performed the combined analysis of massively parallel whole-genome and transcriptome sequencing for cancer and paired normal tissue of a 33-yr-old lung adenocarcinoma patient, who is a never-smoker and has no familial cancer history. The cancer showed no known driver mutation in EGFR or KRAS and no EML4-ALK fusion. Here we report a novel fusion gene between KIF5B and the RET proto-oncogene caused by a pericentric inversion of 10p11.22-q11.21. This fusion gene overexpresses chimeric RET receptor tyrosine kinase, which could spontaneously induce cellular transformation. We identified the KIF5B-RET fusion in two more cases out of 20 primary lung adenocarcinomas in the replication study. Our data demonstrate that a subset of NSCLCs could be caused by a fusion of KIF5B and RET, and suggest the chimeric oncogene as a promising molecular target for the personalized diagnosis and treatment of lung cancer.

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Promoter architecture of mouse olfactory receptor genes.

Publication Date: 2012 Jan 24 PMID: 22194471
Authors: Plessy, C. - Pascarella, G. - Bertin, N. - Akalin, A. - Carrieri, C. - Vassalli, A. - Lazarevic, D. - Severin, J. - Vlachouli, C. - Simone, R. - Faulkner, G. J. - Kawai, J. - Daub, C. O. - Zucchelli, S. - Hayashizaki, Y. - Mombaerts, P. - Lenhard, B. - Gustincich, S. - Carninci, P.
Journal: Genome Res

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

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The human phosphotyrosine signaling network: Evolution and hotspots of hijacking in cancer.

Publication Date: 2012 Jan 26 PMID: 22194470
Authors: Li, L. - Tibiche, C. - Fu, C. - Kaneko, T. - Moran, M. F. - Schiller, M. R. - Li, S. S. - Wang, E.
Journal: Genome Res

Phosphotyrosine (pTyr) signaling, which plays a central role in cell-cell and cell-environment interactions, has been considered to be an evolutionary innovation in multicellular metazoans. However, neither the emergence nor the evolution of the human pTyr signaling system is currently understood. Tyrosine kinase (TK) circuits, each of which consists of a TK writer, a kinase substrate, and a related reader, such as Src homology (SH) 2 domains and pTyr-binding (PTB) domains, comprise the core machinery of the pTyr signaling network. In this study, we analyzed the evolutionary trajectories of 583 literature-derived and 50,000 computationally predicted human TK circuits in 19 representative eukaryotic species and assigned their evolutionary origins. We found that human TK circuits for intracellular pTyr signaling originated largely from primitive organisms, whereas the inter- or extracellular signaling circuits experienced significant expansion in the bilaterian lineage through the "back-wiring" of newly evolved kinases to primitive substrates and SH2/PTB domains. Conversely, the TK circuits that are involved in tissue-specific signaling evolved mainly in vertebrates by the back-wiring of vertebrate substrates to primitive kinases and SH2/PTB domains. Importantly, we found that cancer signaling preferentially employs the pTyr sites, which are linked to more TK circuits. Our work provides insights into the evolutionary paths of the human pTyr signaling circuits and suggests the use of a network approach for cancer intervention through the targeting of key pTyr sites and their associated signaling hubs in the network.

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Whole genome sequencing of matched primary and metastatic acral melanomas.

Publication Date: 2012 Feb PMID: 22183965
Authors: Turajlic, S. - Furney, S. J. - Lambros, M. B. - Mitsopoulos, C. - Kozarewa, I. - Geyer, F. C. - Mackay, A. - Hakas, J. - Zvelebil, M. - Lord, C. J. - Ashworth, A. - Thomas, M. - Stamp, G. - Larkin, J. - Reis-Filho, J. S. - Marais, R.
Journal: Genome Res

Next generation sequencing has enabled systematic discovery of mutational spectra in cancer samples. Here, we used whole genome sequencing to characterize somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Our data show that the somatic mutational rates in this acral melanoma sample pair were more comparable to the rates reported in cancer genomes not associated with mutagenic exposure than in the genome of a melanoma cell line or the transcriptome of melanoma short-term cultures. Despite the perception that acral skin is sun-protected, the dominant mutational signature in these samples is compatible with damage due to ultraviolet light exposure. A nonsense mutation in ERCC5 discovered in both the primary and metastatic tumors could also have contributed to the mutational signature through accumulation of unrepaired dipyrimidine lesions. However, evidence of transcription-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. The primary and the metastasis are highly similar at the level of global gene copy number alterations, loss of heterozygosity and single nucleotide variation (SNV). Furthermore, the majority of the SNVs in the primary tumor were propagated in the metastasis and one nonsynonymous coding SNV and one splice site mutation appeared to arise de novo in the metastatic lesion.

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Cooperation between Polycomb and androgen receptor during oncogenic transformation.

Publication Date: 2012 Feb PMID: 22179855
Authors: Zhao, J. C. - Yu, J. - Runkle, C. - Wu, L. - Hu, M. - Wu, D. - Liu, J. S. - Wang, Q. - Qin, Z. S. - Yu, J.
Journal: Genome Res

Androgen receptor (AR) is a hormone-activated transcription factor that plays important roles in prostate development and function, as well as malignant transformation. The downstream pathways of AR, however, are incompletely understood. AR has been primarily known as a transcriptional activator inducing prostate-specific gene expression. Through integrative analysis of genome-wide AR occupancy and androgen-regulated gene expression, here we report AR as a globally acting transcriptional repressor. This repression is mediated by androgen-responsive elements (ARE) and dictated by Polycomb group protein EZH2 and repressive chromatin remodeling. In embryonic stem cells, AR-repressed genes are occupied by EZH2 and harbor bivalent H3K4me3 and H3K27me3 modifications that are characteristic of differentiation regulators, the silencing of which maintains the undifferentiated state. Concordantly, these genes are silenced in castration-resistant prostate cancer rendering a stem cell-like lack of differentiation and tumor progression. Collectively, our data reveal an unexpected role of AR as a transcriptional repressor inhibiting non-prostatic differentiation and, upon excessive signaling, resulting in cancerous dedifferentiation.

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Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

Publication Date: 2012 Feb PMID: 22156296
Authors: Hon, G. C. - Hawkins, R. D. - Caballero, O. L. - Lo, C. - Lister, R. - Pelizzola, M. - Valsesia, A. - Ye, Z. - Kuan, S. - Edsall, L. E. - Camargo, A. A. - Stevenson, B. J. - Ecker, J. R. - Bafna, V. - Strausberg, R. L. - Simpson, A. J. - Ren, B.
Journal: Genome Res

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

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Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency.

Publication Date: 2012 Feb PMID: 22156295
Authors: Wang, L. - Tsutsumi, S. - Kawaguchi, T. - Nagasaki, K. - Tatsuno, K. - Yamamoto, S. - Sang, F. - Sonoda, K. - Sugawara, M. - Saiura, A. - Hirono, S. - Yamaue, H. - Miki, Y. - Isomura, M. - Totoki, Y. - Nagae, G. - Isagawa, T. - Ueda, H. - Murayama-Hosokawa, S. - Shibata, T. - Sakamoto, H. - Kanai, Y. - Kaneda, A. - Noda, T. - Aburatani, H.
Journal: Genome Res

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

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Efficient de novo assembly of large genomes using compressed data structures.

Publication Date: 2012 Jan 23 PMID: 22156294
Authors: Simpson, J. T. - Durbin, R.
Journal: Genome Res

De novo genome sequence assembly is important both to generate new sequence assemblies for previously uncharacterized genomes and to identify the genome sequence of individuals in a reference-unbiased way. We present memory efficient data structures and algorithms for assembly using the FM-index derived from the compressed Burrows-Wheeler transform, and a new assembler based on these called SGA (String Graph Assembler). We describe algorithms to error-correct, assemble, and scaffold large sets of sequence data. SGA uses the overlap-based string graph model of assembly, unlike most de novo assemblers that rely on de Bruijn graphs, and is simply parallelizable. We demonstrate the error correction and assembly performance of SGA on 1.2 billion sequence reads from a human genome, which we are able to assemble using 54 GB of memory. The resulting contigs are highly accurate and contiguous, while covering 95% of the reference genome (excluding contigs <200 bp in length). Because of the low memory requirements and parallelization without requiring inter-process communication, SGA provides the first practical assembler to our knowledge for a mammalian-sized genome on a low-end computing cluster.

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GAGE: A critical evaluation of genome assemblies and assembly algorithms.

Publication Date: 2012 Jan 12 PMID: 22147368
Authors: Salzberg, S. L. - Phillippy, A. M. - Zimin, A. - Puiu, D. - Magoc, T. - Koren, S. - Treangen, T. J. - Schatz, M. C. - Delcher, A. L. - Roberts, M. - Marcais, G. - Pop, M. - Yorke, J. A.
Journal: Genome Res

New sequencing technology has dramatically altered the landscape of whole-genome sequencing, allowing scientists to initiate numerous projects to decode the genomes of previously unsequenced organisms. The lowest-cost technology can generate deep coverage of most species, including mammals, in just a few days. The sequence data generated by one of these projects consist of millions or billions of short DNA sequences (reads) that range from 50 to 150 nt in length. These sequences must then be assembled de novo before most genome analyses can begin. Unfortunately, genome assembly remains a very difficult problem, made more difficult by shorter reads and unreliable long-range linking information. In this study, we evaluated several of the leading de novo assembly algorithms on four different short-read data sets, all generated by Illumina sequencers. Our results describe the relative performance of the different assemblers as well as other significant differences in assembly difficulty that appear to be inherent in the genomes themselves. Three overarching conclusions are apparent: first, that data quality, rather than the assembler itself, has a dramatic effect on the quality of an assembled genome; second, that the degree of contiguity of an assembly varies enormously among different assemblers and different genomes; and third, that the correctness of an assembly also varies widely and is not well correlated with statistics on contiguity. To enable others to replicate our results, all of our data and methods are freely available, as are all assemblers used in this study.

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Disclosing pathogenic genetic variants to research participants: Quantifying an emerging ethical responsibility.

Publication Date: 2012 Jan 9 PMID: 22147367
Authors: Cassa, C. A. - Savage, S. K. - Taylor, P. L. - Green, R. C. - McGuire, A. L. - Mandl, K. D.
Journal: Genome Res

There is an emerging consensus that when investigators obtain genomic data from research participants, they may incur an ethical responsibility to inform at-risk individuals about clinically significant variants discovered during the course of their research. With whole-exome sequencing becoming commonplace and the falling costs of full-genome sequencing, there will be an increasingly large number of variants identified in research participants that may be of sufficient clinical relevance to share. An explicit approach to triaging and communicating these results has yet to be developed, and even the magnitude of the task is uncertain. To develop an estimate of the number of variants that might qualify for disclosure, we apply recently published recommendations for the return of results to a defined and representative set of variants and then extrapolate these estimates to genome scale. We find that the total number of variants meeting the threshold for recommended disclosure ranges from 3955-12,579 (3.79%-12.06%, 95% CI) in the most conservative estimate to 6998-17,189 (6.69%-16.48%, 95% CI) in an estimate including variants with variable disease expressivity. Additionally, if the growth rate from the previous 4 yr continues, we estimate that the total number of disease-associated variants will grow 37% over the next 4 yr.

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Sequence shortening in the rodent ancestor.

Publication Date: 2012 Jan 9 PMID: 22128134
Authors: Laurie, S. - Toll-Riera, M. - Rado-Trilla, N. - Alba, M. M.
Journal: Genome Res

Insertions and deletions (indels), together with nucleotide substitutions, are major drivers of sequence evolution. An excess of deletions over insertions in genomic sequences-the so-called deletional bias-has been reported in a wide range of species, including mammals. However, this bias has not been found in the coding sequences of some mammalian species, such as human and mouse. To determine the strength of the deletional bias in mammals, and the influence of mutation and selection, we have quantified indels in both neutrally evolving noncoding sequences and protein-coding sequences, in six mammalian branches: human, macaque, ancestral primate, mouse, rat, and ancestral rodent. The results obtained with an improved algorithm for the placement of insertions in multiple alignments, Prank(+F), indicate that contrary to previous results, the only mammalian branch with a strong deletional bias is the rodent ancestral branch. We estimate that such a bias has resulted in an approximately 2.5% sequence loss of mammalian syntenic region in the ancestor of the mouse and rat. Further, a comparison of coding and noncoding sequences shows that negative selection is acting more strongly against mutations generating amino acid insertions than against mutations resulting in amino acid deletions. The strength of selection against indels is found to be higher in the rodent branches than in the primate branches, consistent with the larger effective population sizes of the rodents.

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Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis.

Publication Date: 2012 Jan 9 PMID: 22110045
Authors: Pauli, A. - Valen, E. - Lin, M. F. - Garber, M. - Vastenhouw, N. L. - Levin, J. Z. - Fan, L. - Sandelin, A. - Rinn, J. L. - Regev, A. - Schier, A. F.
Journal: Genome Res

Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.

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Calling amplified haplotypes in next generation tumor sequence data.

Publication Date: 2012 Feb PMID: 22090379
Authors: Dewal, N. - Hu, Y. - Freedman, M. L. - Laframboise, T. - Pe'er, I.
Journal: Genome Res

During tumor initiation and progression, cancer cells acquire a selective advantage, allowing them to outcompete their normal counterparts. Identification of the genetic changes that underlie these tumor acquired traits can provide deeper insights into the biology of tumorigenesis. Regions of copy number alterations and germline DNA variants are some of the elements subject to selection during tumor evolution. Integrated examination of inherited variation and somatic alterations holds the potential to reveal specific nucleotide alleles that a tumor "prefers" to have amplified. Next-generation sequencing of tumor and matched normal tissues provides a high-resolution platform to identify and analyze such somatic amplicons. Within an amplicon, examination of informative (e.g., heterozygous) sites deviating from a 1:1 ratio may suggest selection of that allele. A naive approach examines the reads for each heterozygous site in isolation; however, this ignores available valuable linkage information across sites. We, therefore, present a novel hidden Markov model-based method-Haplotype Amplification in Tumor Sequences (HATS)-that analyzes tumor and normal sequence data, along with training data for phasing purposes, to infer amplified alleles and haplotypes in regions of copy number gain. Our method is designed to handle rare variants and biases in read data. We assess the performance of HATS using simulated amplified regions generated from varying copy number and coverage levels, followed by amplicons in real data. We demonstrate that HATS infers the amplified alleles more accurately than does the naive approach, especially at low to intermediate coverage levels and in cases (including high coverage) possessing stromal contamination or allelic bias.

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Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription.

Publication Date: 2012 Feb PMID: 22086061
Authors: Patel, M. - Simon, J. M. - Iglesia, M. D. - Wu, S. B. - McFadden, A. W. - Lieb, J. D. - Davis, I. J.
Journal: Genome Res

Chromosomal translocations involving transcription factor genes have been identified in an increasingly wide range of cancers. Some translocations can create a protein "chimera" that is composed of parts from different proteins. How such chimeras cause cancer, and why they cause cancer in some cell types but not others, is not understood. One such chimera is EWS-FLI, the most frequently occurring translocation in Ewing Sarcoma, a malignant bone and soft tissue tumor of children and young adults. Using EWS-FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS-FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS-FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS-FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS-FLI acquired the ability to alter chromatin. Expression of EWS-FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS-FLI in tumor cells restored nucleosome occupancy. Thus, the EWS-FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately, transcriptional dysregulation.

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Genomic analysis identifies association of Fusobacterium with colorectal carcinoma.

Publication Date: 2012 Feb PMID: 22009990
Authors: Kostic, A. D. - Gevers, D. - Pedamallu, C. S. - Michaud, M. - Duke, F. - Earl, A. M. - Ojesina, A. I. - Jung, J. - Bass, A. J. - Tabernero, J. - Baselga, J. - Liu, C. - Shivdasani, R. A. - Ogino, S. - Birren, B. W. - Huttenhower, C. - Garrett, W. S. - Meyerson, M.
Journal: Genome Res

The tumor microenvironment of colorectal carcinoma is a complex community of genomically altered cancer cells, nonneoplastic cells, and a diverse collection of microorganisms. Each of these components may contribute to carcinogenesis; however, the role of the microbiota is the least well understood. We have characterized the composition of the microbiota in colorectal carcinoma using whole genome sequences from nine tumor/normal pairs. Fusobacterium sequences were enriched in carcinomas, confirmed by quantitative PCR and 16S rDNA sequence analysis of 95 carcinoma/normal DNA pairs, while the Bacteroidetes and Firmicutes phyla were depleted in tumors. Fusobacteria were also visualized within colorectal tumors using FISH. These findings reveal alterations in the colorectal cancer microbiota; however, the precise role of Fusobacteria in colorectal carcinoma pathogenesis requires further investigation.

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Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

Publication Date: 2012 Feb PMID: 22009989
Authors: Castellarin, M. - Warren, R. L. - Freeman, J. D. - Dreolini, L. - Krzywinski, M. - Strauss, J. - Barnes, R. - Watson, P. - Allen-Vercoe, E. - Moore, R. A. - Holt, R. A.
Journal: Genome Res

An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p = 2.5 x 10(-6)), and we observed a positive association with lymph node metastasis.

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Estimation of rearrangement phylogeny for cancer genomes.

Publication Date: 2012 Feb PMID: 21994251
Authors: Greenman, C. D. - Pleasance, E. D. - Newman, S. - Yang, F. - Fu, B. - Nik-Zainal, S. - Jones, D. - Lau, K. W. - Carter, N. - Edwards, P. A. - Futreal, P. A. - Stratton, M. R. - Campbell, P. J.
Journal: Genome Res

Cancer genomes are complex, carrying thousands of somatic mutations including base substitutions, insertions and deletions, rearrangements, and copy number changes that have been acquired over decades. Recently, technologies have been introduced that allow generation of high-resolution, comprehensive catalogs of somatic alterations in cancer genomes. However, analyses of these data sets generally do not indicate the order in which mutations have occurred, or the resulting karyotype. Here, we introduce a mathematical framework that begins to address this problem. By using samples with accurate data sets, we can reconstruct relatively complex temporal sequences of rearrangements and provide an assembly of genomic segments into digital karyotypes. For cancer genes mutated in rearranged regions, this information can provide a chronological examination of the selective events that have taken place.

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Unique DNA methylome profiles in CpG island methylator phenotype colon cancers.

Publication Date: 2012 Feb PMID: 21990380
Authors: Xu, Y. - Hu, B. - Choi, A. J. - Gopalan, B. - Lee, B. H. - Kalady, M. F. - Church, J. M. - Ting, A. H.
Journal: Genome Res

A subset of colorectal cancers was postulated to have the CpG island methylator phenotype (CIMP), a higher propensity for CpG island DNA methylation. The validity of CIMP, its molecular basis, and its prognostic value remain highly controversial. Using MBD-isolated genome sequencing, we mapped and compared genome-wide DNA methylation profiles of normal, non-CIMP, and CIMP colon specimens. Multidimensional scaling analysis revealed that each specimen could be clearly classified as normal, non-CIMP, and CIMP, thus signifying that these three groups have distinctly different global methylation patterns. We discovered 3780 sites in various genomic contexts that were hypermethylated in both non-CIMP and CIMP colon cancers when compared with normal colon. An additional 2026 sites were found to be hypermethylated in CIMP tumors only; and importantly, 80% of these sites were located in CpG islands. These data demonstrate on a genome-wide level that the additional hypermethylation seen in CIMP tumors occurs almost exclusively at CpG islands and support definitively that these tumors were appropriately named. When these sites were examined more closely, we found that 25% were adjacent to sites that were also hypermethylated in non-CIMP tumors. Thus, CIMP is also characterized by more extensive methylation of sites that are already prone to be hypermethylated in colon cancer. These observations indicate that CIMP tumors have specific defects in controlling both DNA methylation seeding and spreading and serve as an important first step in delineating molecular mechanisms that control these processes.

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Genomic analysis of circulating cell-free DNA infers breast cancer dormancy.

Publication Date: 2012 Feb PMID: 21990379
Authors: Shaw, J. A. - Page, K. - Blighe, K. - Hava, N. - Guttery, D. - Ward, B. - Brown, J. - Ruangpratheep, C. - Stebbing, J. - Payne, R. - Palmieri, C. - Cleator, S. - Walker, R. A. - Coombes, R. C.
Journal: Genome Res

Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cfDNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cfDNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cfDNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cfDNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cfDNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cfDNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cfDNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cfDNA infer dormancy/minimal residual disease in the majority of patients on follow-up.

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Integrating genetic and gene expression evidence into genome-wide association analysis of gene sets.

Publication Date: 2012 Feb PMID: 21940837
Authors: Xiong, Q. - Ancona, N. - Hauser, E. R. - Mukherjee, S. - Furey, T. S.
Journal: Genome Res

Single variant or single gene analyses generally account for only a small proportion of the phenotypic variation in complex traits. Alternatively, gene set or pathway association analyses are playing an increasingly important role in uncovering genetic architectures of complex traits through the identification of systematic genetic interactions. Two dominant paradigms for gene set analyses are association analyses based on SNP genotypes and those based on gene expression profiles. However, gene-disease association can manifest in many ways, such as alterations of gene expression, genotype, and copy number; thus, an integrative approach combining multiple forms of evidence can more accurately and comprehensively capture pathway associations. We have developed a single statistical framework, Gene Set Association Analysis (GSAA), that simultaneously measures genome-wide patterns of genetic variation and gene expression variation to identify sets of genes enriched for differential expression and/or trait-associated genetic markers. Simulation studies illustrate that joint analyses of genomic data increase the power to detect real associations when compared with gene set methods that use only one genomic data type. The analysis of two human diseases, glioblastoma and Crohn's disease, detected abnormalities in previously identified disease-associated pathways, such as pathways related to PI3K signaling, DNA damage response, and the activation of NFKB. In addition, GSAA predicted novel pathway associations, for example, differential genetic and expression characteristics in genes from the ABC transporter family in glioblastoma and from the HLA system in Crohn's disease. These demonstrate that GSAA can help uncover biological pathways underlying human diseases and complex traits.

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Mutual exclusivity analysis identifies oncogenic network modules.

Publication Date: 2012 Feb PMID: 21908773
Authors: Ciriello, G. - Cerami, E. - Sander, C. - Schultz, N.
Journal: Genome Res

Although individual tumors of the same clinical type have surprisingly diverse genomic alterations, these events tend to occur in a limited number of pathways, and alterations that affect the same pathway tend to not co-occur in the same patient. While pathway analysis has been a powerful tool in cancer genomics, our knowledge of oncogenic pathway modules is incomplete. To systematically identify such modules, we have developed a novel method, Mutual Exclusivity Modules in cancer (MEMo). The method uses correlation analysis and statistical tests to identify network modules by three criteria: (1) Member genes are recurrently altered across a set of tumor samples; (2) member genes are known to or are likely to participate in the same biological process; and (3) alteration events within the modules are mutually exclusive. Applied to data from the Cancer Genome Atlas (TCGA), the method identifies the principal known altered modules in glioblastoma (GBM) and highlights the striking mutual exclusivity of genomic alterations in the PI(3)K, p53, and Rb pathways. In serous ovarian cancer, we make the novel observation that inactivation of BRCA1 and BRCA2 is mutually exclusive of amplification of CCNE1 and inactivation of RB1, suggesting distinct alternative causes of genomic instability in this cancer type; and, we identify RBBP8 as a candidate oncogene involved in Rb-mediated cell cycle control. When applied to any cancer genomics data set, the algorithm can nominate oncogenic alterations that have a particularly strong selective effect and may also be useful in the design of therapeutic combinations in cases where mutual exclusivity reflects synthetic lethality.

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Genetic variation in radiation-induced cell death.

Publication Date: 2012 Feb PMID: 21844125
Authors: Smirnov, D. A. - Brady, L. - Halasa, K. - Morley, M. - Solomon, S. - Cheung, V. G.
Journal: Genome Res

Radiation exposure through environmental, medical, and occupational settings is increasingly common. While radiation has harmful effects, it has utility in many applications such as radiotherapy for cancer. To increase the efficacy of radiation treatment and minimize its risks, a better understanding of the individual differences in radiosensitivity and the molecular basis of radiation response is needed. Here, we integrated human genetic and functional genomic approaches to study the response of human cells to radiation. We measured radiation-induced changes in gene expression and cell death in B cells from normal individuals. We found extensive individual variation in gene expression and cellular responses. To understand the genetic basis of this variation, we mapped the DNA sequence variants that influence expression response to radiation. We also identified radiation-responsive genes that regulate cell death; silencing of these genes by small interfering RNA led to an increase in radiation-induced cell death in human B cells, colorectal and prostate cancer cells. Together these results uncovered DNA variants that contribute to radiosensitivity and identified genes that can be targeted to increase the sensitivity of tumors to radiation.

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Acetylation of H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic remodeling in cancer.

Publication Date: 2012 Feb PMID: 21788347
Authors: Valdes-Mora, F. - Song, J. Z. - Statham, A. L. - Strbenac, D. - Robinson, M. D. - Nair, S. S. - Patterson, K. I. - Tremethick, D. J. - Stirzaker, C. - Clark, S. J.
Journal: Genome Res

Histone H2A.Z (H2A.Z) is an evolutionarily conserved H2A variant implicated in the regulation of gene expression; however, its role in transcriptional deregulation in cancer remains poorly understood. Using genome-wide studies, we investigated the role of promoter-associated H2A.Z and acetylated H2A.Z (acH2A.Z) in gene deregulation and its relationship with DNA methylation and H3K27me3 in prostate cancer. Our results reconcile the conflicting reports of positive and negative roles for histone H2A.Z and gene expression states. We find that H2A.Z is enriched in a bimodal distribution at nucleosomes, surrounding the transcription start sites (TSSs) of both active and poised gene promoters. In addition, H2A.Z spreads across the entire promoter of inactive genes in a deacetylated state. In contrast, acH2A.Z is only localized at the TSSs of active genes. Gene deregulation in cancer is also associated with a reorganization of acH2A.Z and H2A.Z nucleosome occupancy across the promoter region and TSS of genes. Notably, in cancer cells we find that a gain of acH2A.Z at the TSS occurs with an overall decrease of H2A.Z levels, in concert with oncogene activation. Furthermore, deacetylation of H2A.Z at TSSs is increased with silencing of tumor suppressor genes. We also demonstrate that acH2A.Z anti-correlates with promoter H3K27me3 and DNA methylation. We show for the first time, that acetylation of H2A.Z is a key modification associated with gene activity in normal cells and epigenetic gene deregulation in tumorigenesis.

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Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations.

Publication Date: 2012 Feb PMID: 21765009
Authors: Thompson, J. F. - Reifenberger, J. G. - Giladi, E. - Kerouac, K. - Gill, J. - Hansen, E. - Kahvejian, A. - Kapranov, P. - Knope, T. - Lipson, D. - Steinmann, K. E. - Milos, P. M.
Journal: Genome Res

Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.

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Assessment of palindromes as platforms for DNA amplification in breast cancer.

Publication Date: 2012 Feb PMID: 21752925
Authors: Guenthoer, J. - Diede, S. J. - Tanaka, H. - Chai, X. - Hsu, L. - Tapscott, S. J. - Porter, P. L.
Journal: Genome Res

DNA amplification, particularly of chromosomes 8 and 11, occurs frequently in breast cancer and is a key factor in tumorigenesis, often associated with poor prognosis. The mechanisms involved in the amplification of these regions are not fully understood. Studies from model systems have demonstrated that palindrome formation can be an early step in DNA amplification, most notably seen in the breakage-fusion-bridge (BFB) cycle. Therefore, palindromes might be associated with gene amplicons in breast cancer. To address this possibility, we coupled high-resolution palindrome profiling by the Genome-wide Analysis of Palindrome Formation (GAPF) assay with genome-wide copy-number analyses on a set of breast cancer cell lines and primary tumors to spatially associate palindromes and copy-number gains. We identified GAPF-positive regions distributed nonrandomly throughout cell line and tumor genomes, often in clusters, and associated with copy-number gains. Commonly amplified regions in breast cancer, chromosomes 8q and 11q, had GAPF-positive regions flanking and throughout the copy-number gains. We also identified amplification-associated GAPF-positive regions at similar locations in subsets of breast cancers with similar characteristics (e.g., ERBB2 amplification). These shared positive regions offer the potential to evaluate the utility of palindromes as prognostic markers, particularly in premalignant breast lesions. Our results implicate palindrome formation in the amplification of regions with key roles in breast tumorigenesis, particularly in subsets of breast cancers.

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Genome-scale analysis of aberrant DNA methylation in colorectal cancer.

Publication Date: 2012 Feb PMID: 21659424
Authors: Hinoue, T. - Weisenberger, D. J. - Lange, C. P. - Shen, H. - Byun, H. M. - Van Den Berg, D. - Malik, S. - Pan, F. - Noushmehr, H. - van Dijk, C. M. - Tollenaar, R. A. - Laird, P. W.
Journal: Genome Res

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation-based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAF(V600E) mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent approximately 7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing.

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De novo discovery of mutated driver pathways in cancer.

Publication Date: 2012 Feb PMID: 21653252
Authors: Vandin, F. - Upfal, E. - Raphael, B. J.
Journal: Genome Res

Next-generation DNA sequencing technologies are enabling genome-wide measurements of somatic mutations in large numbers of cancer patients. A major challenge in the interpretation of these data is to distinguish functional "driver mutations" important for cancer development from random "passenger mutations." A common approach for identifying driver mutations is to find genes that are mutated at significant frequency in a large cohort of cancer genomes. This approach is confounded by the observation that driver mutations target multiple cellular signaling and regulatory pathways. Thus, each cancer patient may exhibit a different combination of mutations that are sufficient to perturb these pathways. This mutational heterogeneity presents a problem for predicting driver mutations solely from their frequency of occurrence. We introduce two combinatorial properties, coverage and exclusivity, that distinguish driver pathways, or groups of genes containing driver mutations, from groups of genes with passenger mutations. We derive two algorithms, called Dendrix, to find driver pathways de novo from somatic mutation data. We apply Dendrix to analyze somatic mutation data from 623 genes in 188 lung adenocarcinoma patients, 601 genes in 84 glioblastoma patients, and 238 known mutations in 1000 patients with various cancers. In all data sets, we find groups of genes that are mutated in large subsets of patients and whose mutations are approximately exclusive. Our Dendrix algorithms scale to whole-genome analysis of thousands of patients and thus will prove useful for larger data sets to come from The Cancer Genome Atlas (TCGA) and other large-scale cancer genome sequencing projects.

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A DNA methylation fingerprint of 1628 human samples.

Publication Date: 2012 Feb PMID: 21613409
Authors: Fernandez, A. F. - Assenov, Y. - Martin-Subero, J. I. - Balint, B. - Siebert, R. - Taniguchi, H. - Yamamoto, H. - Hidalgo, M. - Tan, A. C. - Galm, O. - Ferrer, I. - Sanchez-Cespedes, M. - Villanueva, A. - Carmona, J. - Sanchez-Mut, J. V. - Berdasco, M. - Moreno, V. - Capella, G. - Monk, D. - Ballestar, E. - Ropero, S. - Martinez, R. - Sanchez-Carbayo, M. - Prosper, F. - Agirre, X. - Fraga, M. F. - Grana, O. - Perez-Jurado, L. - Mora, J. - Puig, S. - Prat, J. - Badimon, L. - Puca, A. A. - Meltzer, S. J. - Lengauer, T. - Bridgewater, J. - Bock, C. - Esteller, M.
Journal: Genome Res

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

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