Genome Research

Global analyses of UPF1 binding and function reveals expanded scope of nonsense-mediated mRNA decay.

Publication Date: 2013 Jun 13 PMID: 23766421
Authors: Hurt, J. A. - Robertson, A. D. - Burge, C. B.
Journal: Genome Res

UPF1 is a DNA/RNA helicase with essential roles in nonsense-mediated mRNA decay (NMD) and embryonic development. How UPF1 regulates target abundance and the relationship between NMD and embryogenesis are not well understood. To explore how NMD shapes the embryonic transcriptome, we integrated genome-wide analyses of UPF1 binding locations, NMD-regulated gene expression, and translation in murine embryonic stem cells (mESCs). We identified over 200 direct UPF1 binding targets using crosslinking/immunoprecipitation-sequencing (CLIP-seq) and revealed a repression pathway that involves 3' UTR binding by UPF1 and translation but is independent of canonical targeting features involving 3' UTR length and stop codon placement. Interestingly, NMD targeting of this set of mRNAs occurs in other mouse tissues and is conserved to human. We also show using ribosome footprint profiling that actively translated upstream open reading frames (uORFs) are enriched in transcription factor mRNAs and predict mRNA repression by NMD, while poorly translated mRNAs escape repression. Together, our results identify novel NMD determinants and targets and provide context for understanding the impact of UPF1 and NMD on the mESC transcriptome.

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Network properties derived from deep sequencing of the human B-cell receptor repertoires delineates B-cell populations.

Publication Date: 2013 Jun 6 PMID: 23742949
Authors: Bashford-Rogers, R. - Palser, A. - Huntly, B. - Rance, R. - Vassiliou, G. - Follows, G. - Kellam, P.
Journal: Genome Res

The adaptive immune response selectively expands B- and T-cell clones following antigen recognition by B- and T-cell receptors (BCR and TCR) respectively. Next-generation sequencing is a powerful tool for dissecting the BCR and TCR populations at high-resolution, but robust computational analyses are required to interpret such sequencing. Here, we develop a novel computational approach for BCR repertoire analysis using established next-generation sequencing methods coupled with network construction and population analysis. BCR sequences organize into networks based on sequence diversity, with differences in network connectivity clearly distinguishing between diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with chronic lymphocytic leukemia (CLL) and other clonal blood disorders. Network population measures defined by Gini Index and cluster sizes quantify the BCR clonality status and are robust to sampling and sequencing depths. BCR network analysis therefore allows the direct and quantifiable comparison of BCR repertoires between samples and intra-individual population changes between temporal or spatially separated samples and over the course of therapy.

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The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA.

Publication Date: 2013 Jun 5 PMID: 23739895
Authors: Regulski, M. - Lu, Z. - Kendall, J. - Donoghue, M. T. - Reinders, J. - Llaca, V. - Deschamps, S. - Smith, A. - Levy, D. - McCombie, W. R. - Tingey, S. - Rafalski, A. - Hicks, J. - Ware, D. - Martienssen, R.
Journal: Genome Res

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24nt, but not 21nt, small interfering RNA (siRNA). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to 8 generations suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.

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Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population.

Publication Date: 2013 Jun 5 PMID: 23739894
Authors: Schmitz, R. J. - He, Y. - Valdes-Lopez, O. - Khan, S. M. - Joshi, T. - Urich, M. A. - Nery, J. R. - Diers, B. - Xu, D. - Stacey, G. - Ecker, J. R.
Journal: Genome Res

Cytosine DNA methylation is one avenue for passing information through cell divisions. Here, we present epigenomic analyses of soybean recombinant inbred lines (RILs) and their parents. Identification of differentially methylated regions (DMRs) revealed DMRs mostly co-segregated with the genotype from which they were derived, but examples of uncoupling of genotype and epigenotype were identified. Linkage mapping of methylation states assessed from whole-genome bisulfite sequencing of 83 RILs uncovered widespread evidence for local methylQTL. This epigenomics approach provides a comprehensive study of the patterns and heritability of methylation variants in a complex genetic population over multiple generations paving the way for understanding how methylation variants contribute to phenotypic variation.

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Comprehensive genome- and transcriptome-wide analyses of mutations associated with microsatellite instability in Korean gastric cancers.

Publication Date: 2013 Jun 4 PMID: 23737375
Authors: Yoon, K. - Lee, S. - Han, T. S. - Moon, S. Y. - Yun, S. M. - Kong, S. H. - Jho, S. - Choe, J. - Yu, J. - Lee, H. J. - Park, J. H. - Kim, H. M. - Lee, S. Y. - Park, J. - Kim, W. H. - Bhak, J. - Yang, H. K. - Kim, S. J.
Journal: Genome Res

Microsatellite instability (MSI) is a critical mechanism that drives genetic aberrations in cancer. To identify the entire MS mutation, we performed the first comprehensive genome- and transcriptome-wide analyses of mutations associated with MSI in Korean gastric cancer cell lines and primary tissues. We identified 18,377 MS mutations of five or more repeat nucleotides in coding sequences and untranslated regions of genes, and discovered 139 individual genes whose expression was down-regulated in association with UTR MS mutation. In addition, we found that 90.5% of MS mutations with deletions in gene regions occurred in UTRs. This analysis emphasizes the genetic diversity of MSI-H gastric tumors and provides clues to the mechanistic basis of instability in microsatellite unstable gastric cancers.

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Next-generation sequencing of paired tyrosine kinase inhibitor-sensitive and -resistant EGFR mutant lung cancer cell lines identifies spectrum of DNA changes associated with drug resistance.

Publication Date: 2013 Jun 3 PMID: 23733853
Authors: Jia, P. - Jin, H. - Meador, C. B. - Xia, J. - Ohashi, K. - Liu, L. - Pirazzoli, V. - Dahlman, K. B. - Politi, K. - Michor, F. - Zhao, Z. - Pao, W.
Journal: Genome Res

Somatic mutations in genes encoding kinases are associated with increased sensitivity of some solid tumors to kinase inhibitors, but patients with metastatic cancer eventually develop disease progression. A common method used to model acquired resistance involves culturing parental drug-sensitive cells with increasing concentrations of drug until cells emerge that are resistant. In EGFR mutant lung cancer, this modeling has reliably identified clinically relevant EGFR tyrosine kinase inhibitor (TKI) resistance mechanisms such as the second-site mutation, EGFR T790M, amplification of the gene encoding an alternative kinase, MET, and epithelial-mesenchymal transition (EMT). The full spectrum of DNA changes associated with EGFR TKI acquired resistance remains unknown. Here, we used next-generation sequencing and bioinformatics analysis to characterize mutational changes associated with 4 populations of EGFR mutant drug-sensitive cell lines and 5 matched drug-resistant cell lines. Comparing resistant cells with their parental counterparts, we identified 16-89 coding SNVs/indels that were acquired and 1-27 that were lost; few SNVs/indels were shared across resistant lines. Comparison of two related parental lines revealed no unique coding SNVs/indels, suggesting that the changes in the resistant lines were due to drug selection. When analyzing whole genome sequencing data from one isogenic pair, we found that there was a higher frequency of SNVs in 'constant late' replication timing zones as compared to 'constant early' replication timing zones (chi-squared p-value < 10-5) and an enrichment of SNV frequencies in genomic regions harboring lamina-associated domains compared to the remainder of the nucleus (chi-squared p-value < 10-5). Surprisingly, we observed a higher burden of CNV changes across all resistant lines, and the one line that had an EMT phenotype displayed significantly higher levels of CNV changes than the other lines with acquired resistance. These results demonstrate a framework for studying the evolution of drug-related genetic variants over time and provide the first genome-wide spectrum of mutations associated with the development of cellular drug resistance in an oncogene-addicted cancer. Collectively, the data suggest that CNV changes may play a larger role than previously appreciated in the acquisition of drug resistance and highlight that resistance may be heterogeneous in the context of different tumor cell backgrounds.

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Integrative phenomics reveals insight into the structure of phenotypic diversity in budding yeast.

Publication Date: 2013 May 29 PMID: 23720455
Authors: Skelly, D. A. - Merrihew, G. E. - Riffle, M. - Connelly, C. F. - Kerr, E. O. - Johansson, M. - Jaschob, D. - Graczyk, B. - Shulman, N. J. - Wakefield, J. - Cooper, S. J. - Fields, S. - Noble, W. S. - Muller, E. G. - Davis, T. N. - Dunham, M. J. - Maccoss, M. J. - Akey, J. M.
Journal: Genome Res

To better understand the quantitative characteristics and structure of phenotypic diversity, we measured over 14,000 transcript, protein, metabolite, and morphological traits in 22 genetically diverse strains of Saccharomyces cerevisiae. Over 50% of all measured traits varied significantly across strains (FDR = 5%). The structure of phenotypic correlations is complex, with 85% of all traits significantly correlated with at least one other phenotype (median = 6, maximum = 328). We show how high-dimensional molecular phenomics datasets can be leveraged to accurately predict phenotypic variation between strains, often with greater precision than afforded by DNA sequence information alone. These results provide new insights into the spectrum and structure of phenotypic diversity and the characteristics influencing the ability to accurately predict phenotypes.

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Cell-type, allelic and genetic signatures in the human pancreatic beta cell transcriptome.

Publication Date: 2013 May 28 PMID: 23716500
Authors: Nica, A. C. - Ongen, H. - Irminger, J. C. - Bosco, D. - Berney, T. - Antonarakis, S. E. - Halban, P. A. - Dermitzakis, E. T.
Journal: Genome Res

Elucidating the pathophysiology and molecular attributes of common disorders as well as developing targeted and effective treatments hinges on the study of the relevant cell type and tissues. Pancreatic beta cells within the islets of Langerhans are centrally involved in the pathogenesis of both type 1 and type 2 diabetes. Describing the differentiated state of the human beta cell has been hampered so far by technical (low resolution microarrays) and biological limitations (whole islet preparations rather than isolated beta cells). We circumvent these by deep RNA sequencing of purified beta cells from 11 individuals, presenting here the first characterization of the human beta cell transcriptome. We perform the first comparison of gene expression profiles between beta cells, whole islets and beta cell depleted islet preparations, revealing thus beta cell specific expression and splicing signatures. Further, we demonstrate that genes with consistent increased expression in beta cells have neuronal-like properties, a signal previously hypothesized. Finally, we find evidence for extensive allelic imbalance in expression and uncover genetic regulatory variants (eQTLs) active in beta cells. This first molecular blueprint of the human beta cell offers biological insight into its differentiated function including expression of key genes associated with both major types of diabetes.

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The genomic landscape of cohesin-associated chromatin interactions.

Publication Date: 2013 May 23 PMID: 23704192
Authors: Demare, L. E. - Leng, J. - Cotney, J. - Reilly, S. K. - Yin, J. - Sarro, R. - Noonan, J. P.
Journal: Genome Res

Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2,264 SMC1A interactions, of which 1,491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue-specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues, but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin-associated interactions in the genome and highlight their role in establishing the regulatory architecture of development.

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DNase-seq predicts regions of rotational nucleosome stability across diverse human cell types.

Publication Date: 2013 Jun 6 PMID: 23657885
Authors: Winter, D. R. - Song, L. - Mukherjee, S. - Furey, T. S. - Crawford, G. E.
Journal: Genome Res

DNase-seq is primarily used to identify nucleosome-depleted DNase I hypersensitive (DHS) sites genome-wide that correspond to active regulatory elements. However, approximately 40 yr ago it was demonstrated that DNase I also digests with a approximately 10-bp periodicity around nucleosomes matching the exposure of the DNA minor groove as it wraps around histones. Here, we use DNase-seq data from 49 samples representing diverse cell types to reveal this digestion pattern at individual loci and predict genomic locations where nucleosome rotational positioning, the orientation of DNA with respect to the histone surface, is stably maintained. We call these regions DNase I annotated regions of nucleosome stability (DARNS). Compared to MNase-seq experiments, we show DARNS correspond well to annotated nucleosomes. Interestingly, many DARNS are positioned over only one side of annotated nucleosomes, suggesting that the periodic digestion pattern attenuates over the nucleosome dyad. DARNS reproduce the arrangement of nucleosomes around transcription start sites and are depleted at ubiquitous DHS sites. We also generated DARNS from multiple lymphoblast cell line (LCL) samples. We found that LCL DARNS were enriched at DHS sites present in most of the original 49 samples but absent in LCLs, while multi-cell-type DARNS were enriched at LCL-specific DHS sites. This indicates that variably open DHS sites are often occupied by rotationally stable nucleosomes in cell types where the DHS site is closed. DARNS provide additional information about precise DNA orientation within individual nucleosomes not available from other nucleosome positioning assays and contribute to understanding the role of chromatin in gene regulation.

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Arboretum: Reconstruction and analysis of the evolutionary history of condition-specific transcriptional modules.

Publication Date: 2013 Jun PMID: 23640720
Authors: Roy, S. - Wapinski, I. - Pfiffner, J. - French, C. - Socha, A. - Konieczka, J. - Habib, N. - Kellis, M. - Thompson, D. - Regev, A.
Journal: Genome Res

Comparative functional genomics studies the evolution of biological processes by analyzing functional data, such as gene expression profiles, across species. A major challenge is to compare profiles collected in a complex phylogeny. Here, we present Arboretum, a novel scalable computational algorithm that integrates expression data from multiple species with species and gene phylogenies to infer modules of coexpressed genes in extant species and their evolutionary histories. We also develop new, generally applicable measures of conservation and divergence in gene regulatory modules to assess the impact of changes in gene content and expression on module evolution. We used Arboretum to study the evolution of the transcriptional response to heat shock in eight species of Ascomycota fungi and to reconstruct modules of the ancestral environmental stress response (ESR). We found substantial conservation in the stress response across species and in the reconstructed components of the ancestral ESR modules. The greatest divergence was in the most induced stress, primarily through module expansion. The divergence of the heat stress response exceeds that observed in the response to glucose depletion in the same species. Arboretum and its associated analyses provide a comprehensive framework to systematically study regulatory evolution of condition-specific responses.

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A broadly applicable high-throughput screening strategy identifies new regulators of Dlg4 (Psd-95) alternative splicing.

Publication Date: 2013 Jun PMID: 23636947
Authors: Zheng, S. - Damoiseaux, R. - Chen, L. - Black, D. L.
Journal: Genome Res

Most mammalian genes produce multiple mRNA isoforms derived from alternative pre-mRNA splicing, with each alternative exon controlled by a complex network of regulatory factors. The identification of these regulators can be laborious and is usually carried out one factor at a time. We have developed a broadly applicable high-throughput screening method that simultaneously identifies multiple positive and negative regulators of a particular exon. Two minigene reporters were constructed: One produces green fluorescent protein (GFP) from the mRNA including an exon, and red fluorescent protein (RFP) from the mRNA lacking the exon; the other switches these fluorescent products of exon inclusion and exclusion. Combining results from these two reporters eliminates many false positives and greatly enriches for true splicing regulators. After extensive optimization of this method, we performed a gain-of-function screen of 15,779 cDNA clones and identified 40 genes affecting exon 18 of Discs large homolog 4 (Dlg4; also known as post-synaptic density protein 95 [Psd-95]). We confirmed that 28 of the 34 recoverable clones alter reporter splicing in RT-PCR assays. Remarkably, 18 of the identified genes encode splicing factors or RNA binding proteins, including PTBP1, a previously identified regulator of this exon. Loss-of-function experiments examining endogenous Dlg4 transcripts validated the effects of five of eight genes tested in independent cell lines, and two genes were further confirmed to regulate Dlg4 splicing in primary neurons. These results identify multiple new regulators of Dlg4 splicing, and validate an approach to isolating splicing regulators for almost any cassette exon from libraries of cDNAs, shRNAs, or small molecules.

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Quantitative proteomic analysis reveals concurrent RNA-protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae.

Publication Date: 2013 Jun PMID: 23636942
Authors: Klass, D. M. - Scheibe, M. - Butter, F. - Hogan, G. J. - Mann, M. - Brown, P. O.
Journal: Genome Res

A growing body of evidence supports the existence of an extensive network of RNA-binding proteins (RBPs) whose combinatorial binding affects the post-transcriptional fate of every mRNA in the cell-yet we still do not have a complete understanding of which proteins bind to mRNA, which of these bind concurrently, and when and where in the cell they bind. We describe here a method to identify the proteins that bind to RNA concurrently with an RBP of interest, using quantitative mass spectrometry combined with RNase treatment of affinity-purified RNA-protein complexes. We applied this method to the known RBPs Pab1, Nab2, and Puf3. Our method significantly enriched for known RBPs and is a clear improvement upon previous approaches in yeast. Our data reveal that some reported protein-protein interactions may instead reflect simultaneous binding to shared RNA targets. We also discovered more than 100 candidate RBPs, and we independently confirmed that 77% (23/30) bind directly to RNA. The previously recognized functions of the confirmed novel RBPs were remarkably diverse, and we mapped the RNA-binding region of one of these proteins, the transcriptional coactivator Mbf1, to a region distinct from its DNA-binding domain. Our results also provided new insights into the roles of Nab2 and Puf3 in post-transcriptional regulation by identifying other RBPs that bind simultaneously to the same mRNAs. While existing methods can identify sets of RBPs that interact with common RNA targets, our approach can determine which of those interactions are concurrent-a crucial distinction for understanding post-transcriptional regulation.

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Mobile element scanning (ME-Scan) identifies thousands of novel Alu insertions in diverse human populations.

Publication Date: 2013 Jun 6 PMID: 23599355
Authors: Witherspoon, D. J. - Zhang, Y. - Xing, J. - Watkins, W. S. - Ha, H. - Batzer, M. A. - Jorde, L. B.
Journal: Genome Res

Alu retrotransposons are the most numerous and active mobile elements in humans, causing genetic disease and creating genomic diversity. Mobile element scanning (ME-Scan) enables comprehensive and affordable identification of mobile element insertions (MEI) using targeted high-throughput sequencing of multiplexed MEI junction libraries. In a single experiment, ME-Scan identifies nearly all AluYb8 and AluYb9 elements, with high sensitivity for both rare and common insertions, in 169 individuals of diverse ancestry. ME-Scan detects heterozygous insertions in single individuals with 91% sensitivity. Insertion presence or absence states determined by ME-Scan are 95% concordant with those determined by locus-specific PCR assays. By sampling diverse populations from Africa, South Asia, and Europe, we are able to identify 5799 Alu insertions, including 2524 novel ones, some of which occur in exons. Sub-Saharan populations and a Pygmy group in particular carry numerous intermediate-frequency Alu insertions that are absent in non-African groups. There is a significant dearth of exon-interrupting insertions among common Alu polymorphisms, but the density of singleton Alu insertions is constant across exonic and nonexonic regions. In one case, a validated novel singleton Alu interrupts a protein-coding exon of FAM187B. This implies that exonic Alu insertions are generally deleterious and thus eliminated by natural selection, but not so quickly that they cannot be observed as extremely rare variants.

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Interplay between chromatin state, regulator binding, and regulatory motifs in six human cell types.

Publication Date: 2013 May 30 PMID: 23595227
Authors: Ernst, J. - Kellis, M.
Journal: Genome Res

The regions bound by sequence-specific transcription factors can be highly variable across different cell types despite the static nature of the underlying genome sequence. This has been partly attributed to changes in chromatin accessibility, but a systematic picture has been hindered by the lack of large-scale data sets. Here, we use 456 binding experiments for 119 regulators and 84 chromatin maps generated by the ENCODE in six human cell types, and relate those to a global map of regulatory motif instances for these factors. We find specific and robust chromatin state preferences for each regulator beyond the previously reported open-chromatin association, suggesting a much richer chromatin landscape beyond simple accessibility. The preferentially bound chromatin states of regulators were enriched for sequence motifs of regulators relative to all states, suggesting that these preferences are at least partly encoded by the genomic sequence. Relative to all regions bound by a regulator, however, regulatory motifs were surprisingly depleted in the regulator's preferentially bound states, suggesting additional non-sequence-specific binding beyond the level predicted by the regulatory motifs. Such permissive binding was largely restricted to open-chromatin regions showing histone modification marks characteristic of active enhancer and promoter regions, whereas open-chromatin regions lacking such marks did not show permissive binding. Lastly, the vast majority of cobinding of regulator pairs is predicted by the chromatin state preferences of individual regulators. Overall, our results suggest a joint role of sequence motifs and specific chromatin states beyond mere accessibility in mediating regulator binding dynamics across different cell types.

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CG methylated microarrays identify a novel methylated sequence bound by the CEBPB|ATF4 heterodimer that is active in vivo.

Publication Date: 2013 Jun PMID: 23590861
Authors: Mann, I. K. - Chatterjee, R. - Zhao, J. - He, X. - Weirauch, M. T. - Hughes, T. R. - Vinson, C.
Journal: Genome Res

To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer occurring three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD, and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. The electrophoretic mobility shift assay (EMSA) confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50x methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TFs, which may be functional in vivo.

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Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing.

Publication Date: 2013 Jun PMID: 23580730
Authors: Arribere, J. A. - Gilbert, W. V.
Journal: Genome Res

Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-sequencing (TL-seq), a technique combining enzymatic capture of m(7)G-capped mRNA 5' ends with high-throughput sequencing. TL-seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Surprisingly, TL-seq identified transcription initiation sites within 6% of protein-coding regions, and these sites were concentrated near the 5' ends of ORFs. Furthermore, ribosome density analysis showed these truncated mRNAs are translated. Translation-associated TL-seq (TATL-seq), which combines TL-seq with polysome fractionation, enabled annotation of TLs, and simultaneously assayed their function in translation. Using TATL-seq to address relationships between TL features and translation of the downstream ORF, we observed that upstream AUGs (uAUGs), and no other upstream codons, were associated with poor translation and nonsense-mediated mRNA decay (NMD). We also identified hundreds of genes with very short TLs, and demonstrated that short TLs were associated with poor translation initiation at the annotated start codon and increased initiation at downstream AUGs. This frequently resulted in out-of-frame translation and subsequent termination at premature termination codons, culminating in NMD of the transcript. Unlike previous approaches, our technique enabled observation of alternative TL variants for hundreds of genes and revealed significant differences in translation in genes with distinct TL isoforms. TL-seq and TATL-seq are useful tools for annotation and functional characterization of TLs, and can be applied to any eukaryotic system to investigate TL-mediated regulation of gene expression.

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Maps of open chromatin highlight cell type-restricted patterns of regulatory sequence variation at hematological trait loci.

Publication Date: 2013 May 24 PMID: 23570689
Authors: Paul, D. S. - Albers, C. A. - Rendon, A. - Voss, K. - Stephens, J. - Consortium, H. - van der Harst, P. - Chambers, J. C. - Soranzo, N. - Ouwehand, W. H. - Deloukas, P.
Journal: Genome Res

Nearly three-quarters of the 143 genetic signals associated with platelet and erythrocyte phenotypes identified by meta-analyses of genome-wide association (GWA) studies are located at non-protein-coding regions. Here, we assessed the role of candidate regulatory variants associated with cell type-restricted, closely related hematological quantitative traits in biologically relevant hematopoietic cell types. We used formaldehyde-assisted isolation of regulatory elements followed by next-generation sequencing (FAIRE-seq) to map regions of open chromatin in three primary human blood cells of the myeloid lineage. In the precursors of platelets and erythrocytes, as well as in monocytes, we found that open chromatin signatures reflect the corresponding hematopoietic lineages of the studied cell types and associate with the cell type-specific gene expression patterns. Dependent on their signal strength, open chromatin regions showed correlation with promoter and enhancer histone marks, distance to the transcription start site, and ontology classes of nearby genes. Cell type-restricted regions of open chromatin were enriched in sequence variants associated with hematological indices. The majority (63.6%) of such candidate functional variants at platelet quantitative trait loci (QTLs) coincided with binding sites of five transcription factors key in regulating megakaryopoiesis. We experimentally tested 13 candidate regulatory variants at 10 platelet QTLs and found that 10 (76.9%) affected protein binding, suggesting that this is a frequent mechanism by which regulatory variants influence quantitative trait levels. Our findings demonstrate that combining large-scale GWA data with open chromatin profiles of relevant cell types can be a powerful means of dissecting the genetic architecture of closely related quantitative traits.

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Cancer translocations in human cells induced by zinc finger and TALE nucleases.

Publication Date: 2013 Jun 6 PMID: 23568838
Authors: Piganeau, M. - Ghezraoui, H. - De Cian, A. - Guittat, L. - Tomishima, M. - Perrouault, L. - Rene, O. - Katibah, G. E. - Zhang, L. - Holmes, M. C. - Doyon, Y. - Concordet, J. P. - Giovannangeli, C. - Jasin, M. - Brunet, E.
Journal: Genome Res

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.

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Genome evolution during progression to breast cancer.

Publication Date: 2013 May 29 PMID: 23568837
Authors: Newburger, D. E. - Kashef-Haghighi, D. - Weng, Z. - Salari, R. - Sweeney, R. T. - Brunner, A. L. - Zhu, S. X. - Guo, X. - Varma, S. - Troxell, M. L. - West, R. B. - Batzoglou, S. - Sidow, A.
Journal: Genome Res

Cancer evolution involves cycles of genomic damage, epigenetic deregulation, and increased cellular proliferation that eventually culminate in the carcinoma phenotype. Early neoplasias, which are often found concurrently with carcinomas and are histologically distinguishable from normal breast tissue, are less advanced in phenotype than carcinomas and are thought to represent precursor stages. To elucidate their role in cancer evolution we performed comparative whole-genome sequencing of early neoplasias, matched normal tissue, and carcinomas from six patients, for a total of 31 samples. By using somatic mutations as lineage markers we built trees that relate the tissue samples within each patient. On the basis of these lineage trees we inferred the order, timing, and rates of genomic events. In four out of six cases, an early neoplasia and the carcinoma share a mutated common ancestor with recurring aneuploidies, and in all six cases evolution accelerated in the carcinoma lineage. Transition spectra of somatic mutations are stable and consistent across cases, suggesting that accumulation of somatic mutations is a result of increased ancestral cell division rather than specific mutational mechanisms. In contrast to highly advanced tumors that are the focus of much of the current cancer genome sequencing, neither the early neoplasia genomes nor the carcinomas are enriched with potentially functional somatic point mutations. Aneuploidies that occur in common ancestors of neoplastic and tumor cells are the earliest events that affect a large number of genes and may predispose breast tissue to eventual development of invasive carcinoma.

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Country-specific antibiotic use practices impact the human gut resistome.

Publication Date: 2013 May 29 PMID: 23568836
Authors: Forslund, K. - Sunagawa, S. - Kultima, J. R. - Mende, D. - Arumugam, M. - Typas, A. - Bork, P.
Journal: Genome Res

Despite increasing concerns over inappropriate use of antibiotics in medicine and food production, population-level resistance transfer into the human gut microbiota has not been demonstrated beyond individual case studies. To determine the "antibiotic resistance potential" for entire microbial communities, we employ metagenomic data and quantify the totality of known resistance genes in each community (its resistome) for 68 classes and subclasses of antibiotics. In 252 fecal metagenomes from three countries, we show that the most abundant resistance determinants are those for antibiotics also used in animals and for antibiotics that have been available longer. Resistance genes are also more abundant in samples from Spain, Italy, and France than from Denmark, the United States, or Japan. Where comparable country-level data on antibiotic use in both humans and animals are available, differences in these statistics match the observed resistance potential differences. The results are robust over time as the antibiotic resistance determinants of individuals persist in the human gut flora for at least a year.

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Genomic insights into the fate of colistin resistance and Acinetobacter baumannii during patient treatment.

Publication Date: 2013 May 29 PMID: 23564252
Authors: Snitkin, E. S. - Zelazny, A. M. - Gupta, J. - Palmore, T. N. - Murray, P. R. - Segre, J. A.
Journal: Genome Res

Bacterial whole-genome sequencing (WGS) of human pathogens has provided unprecedented insights into the evolution of antibiotic resistance. Most studies have focused on identification of resistance mutations, leaving one to speculate on the fate of these mutants once the antibiotic selective pressure is removed. We performed WGS on longitudinal isolates of Acinetobacter baumannii from patients undergoing colistin treatment, and upon subsequent drug withdrawal. In each of the four patients, colistin resistance evolved via mutations at the pmr locus. Upon colistin withdrawal, an ancestral susceptible strain outcompeted resistant isolates in three of the four cases. In the final case, resistance was also lost, but by a compensatory inactivating mutation in the transcriptional regulator of the pmr locus. Notably, this inactivating mutation reduced the probability of reacquiring colistin resistance when subsequently challenged in vitro. On face value, these results supported an in vivo fitness cost preventing the evolution of stable colistin resistance. However, more careful analysis of WGS data identified genomic evidence for stable colistin resistance undetected by clinical microbiological assays. Transcriptional studies validated this genomic hypothesis, showing increased pmr expression of the initial isolate. Moreover, altering the environmental growth conditions of the clinical assay recapitulated the classification as colistin resistant. Additional targeted sequencing revealed that this isolate evolved undetected in a patient undergoing colistin treatment, and was then transmitted to other hospitalized patients, further demonstrating its stability in the absence of colistin. This study provides a unique window into mutational pathways taken in response to antibiotic pressure in vivo, and demonstrates the potential for genome sequence data to predict resistance phenotypes.

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A dynamic H3K27ac signature identifies VEGFA-stimulated endothelial enhancers and requires EP300 activity.

Publication Date: 2013 Jun PMID: 23547170
Authors: Zhang, B. - Day, D. S. - Ho, J. W. - Song, L. - Cao, J. - Christodoulou, D. - Seidman, J. G. - Crawford, G. E. - Park, P. J. - Pu, W. T.
Journal: Genome Res

Histone modifications are now well-established mediators of transcriptional programs that distinguish cell states. However, the kinetics of histone modification and their role in mediating rapid, signal-responsive gene expression changes has been little studied on a genome-wide scale. Vascular endothelial growth factor A (VEGFA), a major regulator of angiogenesis, triggers changes in transcriptional activity of human umbilical vein endothelial cells (HUVECs). Here, we used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to measure genome-wide changes in histone H3 acetylation at lysine 27 (H3K27ac), a marker of active enhancers, in unstimulated HUVECs and HUVECs stimulated with VEGFA for 1, 4, and 12 h. We show that sites with the greatest H3K27ac change upon stimulation were associated tightly with EP300, a histone acetyltransferase. Using the variation of H3K27ac as a novel epigenetic signature, we identified transcriptional regulatory elements that are functionally linked to angiogenesis, participate in rapid VEGFA-stimulated changes in chromatin conformation, and mediate VEGFA-induced transcriptional responses. Dynamic H3K27ac deposition and associated changes in chromatin conformation required EP300 activity instead of altered nucleosome occupancy or changes in DNase I hypersensitivity. EP300 activity was also required for a subset of dynamic H3K27ac sites to loop into proximity of promoters. Our study identified thousands of endothelial, VEGFA-responsive enhancers, demonstrating that an epigenetic signature based on the variation of a chromatin feature is a productive approach to define signal-responsive genomic elements. Further, our study implicates global epigenetic modifications in rapid, signal-responsive transcriptional regulation.

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HCFC1 is a common component of active human CpG-island promoters and coincides with ZNF143, THAP11, YY1, and GABP transcription factor occupancy.

Publication Date: 2013 Jun PMID: 23539139
Authors: Michaud, J. - Praz, V. - James Faresse, N. - Jnbaptiste, C. K. - Tyagi, S. - Schutz, F. - Herr, W.
Journal: Genome Res

In human transcriptional regulation, DNA-sequence-specific factors can associate with intermediaries that orchestrate interactions with a diverse set of chromatin-modifying enzymes. One such intermediary is HCFC1 (also known as HCF-1). HCFC1, first identified in herpes simplex virus transcription, has a poorly defined role in cellular transcriptional regulation. We show here that, in HeLa cells, HCFC1 is observed bound to 5400 generally active CpG-island promoters. Examination of the DNA sequences underlying the HCFC1-binding sites revealed three sequence motifs associated with the binding of (1) ZNF143 and THAP11 (also known as Ronin), (2) GABP, and (3) YY1 sequence-specific transcription factors. Subsequent analysis revealed colocalization of HCFC1 with these four transcription factors at approximately 90% of the 5400 HCFC1-bound promoters. These studies suggest that a relatively small number of transcription factors play a major role in HeLa-cell transcriptional regulation in association with HCFC1.

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Gene expression drives local adaptation in humans.

Publication Date: 2013 May 22 PMID: 23539138
Authors: Fraser, H. B.
Journal: Genome Res

The molecular basis of adaptation-and, in particular, the relative roles of protein-coding versus gene expression changes-has long been the subject of speculation and debate. Recently, the genotyping of diverse human populations has led to the identification of many putative "local adaptations" that differ between populations. Here I show that these local adaptations are over 10-fold more likely to affect gene expression than amino acid sequence. In addition, a novel framework for identifying polygenic local adaptations detects recent positive selection on the expression levels of genes involved in UV radiation response, immune cell proliferation, and diabetes-related pathways. These results provide the first examples of polygenic gene expression adaptation in humans, as well as the first genome-scale support for the hypothesis that changes in gene expression have driven human adaptation.

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Complex expression dynamics and robustness in C. elegans insulin networks.

Publication Date: 2013 Jun PMID: 23539137
Authors: Ritter, A. D. - Shen, Y. - Fuxman Bass, J. - Jeyaraj, S. - Deplancke, B. - Mukhopadhyay, A. - Xu, J. - Driscoll, M. - Tissenbaum, H. A. - Walhout, A. J.
Journal: Genome Res

Gene families expand by gene duplication, and resulting paralogs diverge through mutation. Functional diversification can include neofunctionalization as well as subfunctionalization of ancestral functions. In addition, redundancy in which multiple genes fulfill overlapping functions is often maintained. Here, we use the family of 40 Caenorhabditis elegans insulins to gain insight into the balance between specificity and redundancy. The insulin/insulin-like growth factor (IIS) pathway comprises a single receptor, DAF-2. To date, no single insulin-like peptide recapitulates all DAF-2-associated phenotypes, likely due to redundancy between insulin-like genes. To provide a first-level annotation of potential patterns of redundancy, we comprehensively delineate the spatiotemporal and conditional expression of all 40 insulins in living animals. We observe extensive dynamics in expression that can explain the lack of simple patterns of pairwise redundancy. We propose a model in which gene families evolve to attain differential alliances in different tissues and in response to a range of environmental stresses.

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Integrative analysis of C. elegans modENCODE ChIP-seq data sets to infer gene regulatory interactions.

Publication Date: 2013 Jun PMID: 23531767
Authors: Van Nostrand, E. L. - Kim, S. K.
Journal: Genome Res

The C. elegans modENCODE Consortium has defined in vivo binding sites for a large array of transcription factors by ChIP-seq. In this article, we present examples that illustrate how this compendium of ChIP-seq data can drive biological insights not possible with analysis of individual factors. First, we analyze the number of independent factors bound to the same locus, termed transcription factor complexity, and find that low-complexity sites are more likely to respond to altered expression of a single bound transcription factor. Next, we show that comparison of binding sites for the same factor across developmental stages can reveal insight into the regulatory network of that factor, as we find that the transcription factor UNC-62 has distinct binding profiles at different stages due to distinct cofactor co-association as well as tissue-specific alternative splicing. Finally, we describe an approach to infer potential regulators of gene expression changes found in profiling experiments (such as DNA microarrays) by screening these altered genes to identify significant enrichment for targets of a transcription factor identified in ChIP-seq data sets. After confirming that this approach can correctly identify the upstream regulator on expression data sets for which the regulator was previously known, we applied this approach to identify novel candidate regulators of transcriptional changes with age. The analysis revealed nine candidate aging regulators, of which three were previously known to have a role in longevity. We experimentally showed that two of the new candidate aging regulators can extend lifespan when overexpressed, indicating that this approach can identify novel functional regulators of complex processes.

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Targeted chromosomal deletions and inversions in zebrafish.

Publication Date: 2013 Jun PMID: 23478401
Authors: Gupta, A. - Hall, V. L. - Kok, F. O. - Shin, M. - McNulty, J. C. - Lawson, N. D. - Wolfe, S. A.
Journal: Genome Res

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

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Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants.

Publication Date: 2013 Jun PMID: 23471540
Authors: Enuameh, M. S. - Asriyan, Y. - Richards, A. - Christensen, R. G. - Hall, V. L. - Kazemian, M. - Zhu, C. - Pham, H. - Cheng, Q. - Blatti, C. - Brasefield, J. A. - Basciotta, M. D. - Ou, J. - McNulty, J. C. - Zhu, L. J. - Celniker, S. E. - Sinha, S. - Stormo, G. D. - Brodsky, M. H. - Wolfe, S. A.
Journal: Genome Res

Cys2-His2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes.

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Two DNA-encoded strategies for increasing expression with opposing effects on promoter dynamics and transcriptional noise.

Publication Date: 2013 Jun PMID: 23403035
Authors: Dadiani, M. - van Dijk, D. - Segal, B. - Field, Y. - Ben-Artzi, G. - Raveh-Sadka, T. - Levo, M. - Kaplow, I. - Weinberger, A. - Segal, E.
Journal: Genome Res

Individual cells from a genetically identical population exhibit substantial variation in gene expression. A significant part of this variation is due to noise in the process of transcription that is intrinsic to each gene, and is determined by factors such as the rate with which the promoter transitions between transcriptionally active and inactive states, and the number of transcripts produced during the active state. However, we have a limited understanding of how the DNA sequence affects such promoter dynamics. Here, we used single-cell time-lapse microscopy to compare the effect on transcriptional dynamics of two distinct types of sequence changes in the promoter that can each increase the mean expression of a cell population by similar amounts but through different mechanisms. We show that increasing expression by strengthening a transcription factor binding site results in slower promoter dynamics and higher noise as compared with increasing expression by adding nucleosome-disfavoring sequences. Our results suggest that when achieving the same mean expression, the strategy of using stronger binding sites results in a larger number of transcripts produced from the active state, whereas the strategy of adding nucleosome-disfavoring sequences results in a higher frequency of promoter transitions between active and inactive states. In the latter strategy, this increased sampling of the active state likely reduces the expression variability of the cell population. Our study thus demonstrates the effect of cis-regulatory elements on expression variability and points to concrete types of sequence changes that may allow partial decoupling of expression level and noise.

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Structure-based whole-genome realignment reveals many novel noncoding RNAs.

Publication Date: 2013 Jun PMID: 23296921
Authors: Will, S. - Yu, M. - Berger, B.
Journal: Genome Res

Recent genome-wide computational screens that search for conservation of RNA secondary structure in whole-genome alignments (WGAs) have predicted thousands of structural noncoding RNAs (ncRNAs). The sensitivity of such approaches, however, is limited, due to their reliance on sequence-based whole-genome aligners, which regularly misalign structural ncRNAs. This suggests that many more structural ncRNAs may remain undetected. Structure-based alignment, which could increase the sensitivity, has been prohibitive for genome-wide screens due to its extreme computational costs. Breaking this barrier, we present the pipeline REAPR (RE-Alignment for Prediction of structural ncRNA), which efficiently realigns whole genomes based on RNA sequence and structure, thus allowing us to boost the performance of de novo ncRNA predictors, such as RNAz. Key to the pipeline's efficiency is the development of a novel banding technique for multiple RNA alignment. REAPR significantly outperforms the widely used predictors RNAz and EvoFold in genome-wide screens; in direct comparison to the most recent RNAz screen on D. melanogaster, REAPR predicts twice as many high-confidence ncRNA candidates. Moreover, modENCODE RNA-seq experiments confirm a substantial number of its predictions as transcripts. REAPR's advancement of de novo structural characterization of ncRNAs complements the identification of transcripts from rapidly accumulating RNA-seq data.

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