Browsing by Author "Salzberg, Steven L."
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Item Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments(Genome Biology, 2008-01) Haas, Brian U.; Salzberg, Steven L.; Zhu, Wei; Pertea, Mihaela; Allen, Jonathan E.; Orvis, Joshua; White, Owen; Buell, C Robin; Wortman, Jennifer REVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.Item A clustering method for repeat analysis in DNA sequences.(Genome Biology, 2001-08-01) Volfovsky, Natalia; Haas, Brian J.; Salzberg, Steven L.Background: A computational system for analysis of the repetitive structure of genomic sequences is described. The method uses suffix trees to organize and search the input sequences; this data structure has been used previously for efficient computation of exact and degenerate repeats. Results: The resulting software tool collects all repeat classes and outputs summary statistics as well as a file containing multiple sequences (multi fasta), that can be used as the target of searches. Its use is demonstrated here on several complete microbial genomes, the entire Arabidopsis thaliana genome, and a large collection of rice bacterial artificial chromosome end sequences. Conclusions: We propose a new clustering method for analysis of the repeat data captured in suffix trees. This method has been incorporated into a system that can find repeats in individual genome sequences or sets of sequences, and that can organize those repeats into classes. It quickly and accurately creates repeat databases from small and large genomes. The associated software (RepeatFinder), should prove helpful in the analysis of repeat structure for both complete and partial genome sequences.Item Efficient decoding algorithms for generalized hidden Markov model gene finders(BMC Bioinformatics, 2005-01-24) Majoros, William H.; Pertea, Mihaela; Delcher, Arthur L.; Salzberg, Steven L.Background: The Generalized Hidden Markov Model (GHMM) has proven a useful framework for the task of computational gene prediction in eukaryotic genomes, due to its flexibility and probabilistic underpinnings. As the focus of the gene finding community shifts toward the use of homology information to improve prediction accuracy, extensions to the basic GHMM model are being explored as possible ways to integrate this homology information into the prediction process. Particularly prominent among these extensions are those techniques which call for the simultaneous prediction of genes in two or more genomes at once, thereby increasing significantly the computational cost of prediction and highlighting the importance of speed and memory efficiency in the implementation of the underlying GHMM algorithms. Unfortunately, the task of implementing an efficient GHMM-based gene finder is already a nontrivial one, and it can be expected that this task will only grow more onerous as our models increase in complexity. Results: As a first step toward addressing the implementation challenges of these next-generation systems, we describe in detail two software architectures for GHMM-based gene finders, one comprising the common array-based approach, and the other a highly optimized algorithm which requires significantly less memory while achieving virtually identical speed. We then show how both of these architectures can be accelerated by a factor of two by optimizing their content sensors. We finish with a brief illustration of the impact these optimizations have had on the feasibility of our new homology-based gene finder, TWAIN. Conclusions: In describing a number of optimizations for GHMM-based gene finders and making available two complete open-source software systems embodying these methods, it is our hope that others will be more enabled to explore promising extensions to the GHMM framework, thereby improving the state-of-the-art in gene prediction techniques.Item An empirical analysis of training protocols for probabilistic gene finders(BMC Bioinformatics, 2004-12-21) Majoros, William H.; Salzberg, Steven L.Background: Generalized hidden Markov models (GHMMs) appear to be approaching acceptance as a de facto standard for state-of-the-art ab initio gene finding, as evidenced by the recent proliferation of GHMM implementations. While prevailing methods for modeling and parsing genes using GHMMs have been described in the literature, little attention has been paid as of yet to their proper training. The few hints available in the literature together with anecdotal observations suggest that most practitioners perform maximum likelihood parameter estimation only at the local submodel level, and then attend to the optimization of global parameter structure using some form of ad hoc manual tuning of individual parameters. Results: We decided to investigate the utility of applying a more systematic optimization approach to the tuning of global parameter structure by implementing a global discriminative training procedure for our GHMM-based gene finder. Our results show that significant improvement in prediction accuracy can be achieved by this method. Conclusions: We conclude that training of GHMM-based gene finders is best performed using some form of discriminative training rather than simple maximum likelihood estimation at the submodel level, and that generalized gradient ascent methods are suitable for this task. We also conclude that partitioning of training data for the twin purposes of maximum likelihood initialization and gradient ascent optimization appears to be unnecessary, but that strict segregation of test data must be enforced during final gene finder evaluation to avoid artificially inflated accuracy measurements.Item Evidence for symmetric chromosomal inversions around the replication origin in bacteria(Genome Biology, 2000-12-04) Eisen, Jonathan A.; Heidelberg, John F.; White, Owen; Salzberg, Steven L.Background: Whole-genome comparisons can provide great insight into many aspects of biology. Until recently, however, comparisons were mainly possible only between distantly related species. Complete genome sequences are now becoming available from multiple sets of closely related strains or species. Results: By comparing the recently completed genome sequences of Vibrio cholerae, Streptococcus pneumoniae and Mycobacterium tuberculosis to those of closely related species - Escherichia coli, Streptococcus pyogenes and Mycobacterium leprae, respectively - we have identified an unusual and previously unobserved feature of bacterial genome structure. Scatterplots of the conserved sequences (both DNA and protein) between each pair of species produce a distinct X-shaped pattern, which we call an X-alignment. The key feature of these alignments is that they have symmetry around the replication origin and terminus; that is, the distance of a particular conserved feature (DNA or protein) from the replication origin (or terminus) is conserved between closely related pairs of species. Statistically significant X-alignments are also found within some genomes, indicating that there is symmetry about the replication origin for paralogous features as well. Conclusions: The most likely mechanism of generation of X-alignments involves large chromosomal inversions that reverse the genomic sequence symmetrically around the origin of replication. The finding of these X-alignments between many pairs of species suggests that chromosomal inversions around the origin are a common feature of bacterial genome evolution.Item Full-length messenger RNA sequences greatly improve genome annotation(Genome Biology, 2002-05-30) Haas, Brian J; Volfovsky, Natalia; Town, Christopher D; Troukhan, Maxim; Alexandrov, Nickolai; Feldman, Kenneth A; Flavell, Richard B; White, Owen; Salzberg, Steven L.Background: Annotation of eukaryotic genomes is a complex endeavor that requires the integration of evidence from multiple, often contradictory, sources. With the ever-increasing amount of genome sequence data now available, methods for accurate identification of large numbers of genes have become urgently needed. In an effort to create a set of very high-quality gene models, we used the sequence of 5,000 full-length gene transcripts from Arabidopsis to re-annotate its genome. We have mapped these transcripts to their exact chromosomal locations and, using alignment programs, have created gene models that provide a reference set for this organism. Results: Approximately 35% of the transcripts indicated that previously annotated genes needed modification, and 5% of the transcripts represented newly discovered genes. We also discovered that multiple transcription initiation sites appear to be much more common than previously known, and we report numerous cases of alternative mRNA splicing. We include a comparison of different alignment software and an analysis of how the transcript data improved the previously published annotation. Conclusions: Our results demonstrate that sequencing of large numbers of full-length transcripts followed by computational mapping greatly improves identification of the complete exon structures of eukaryotic genes. In addition, we are able to find numerous introns in the untranslated regions of the genes.Item The Genome Assembly Archive: A New Public Resource(PLoS Biology, 2004-09) Salzberg, Steven L.; Church, Deanna; DiCuccio, Michael; Yaschenko, Eugene; Ostell, JamesItem Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus capsulatus (Bath)(PLoS Biology, 2004-10) Ward, Naomi; Larsen, Øivind; Sakwa, James; Bruseth, Live; Khouri, Hoda; Durkin, A. Scott; Dimitrov, George; Jiang, Lingxia; Scanlan, David; Kang, Katherine H.; Lewis, Matt; Nelson, Karen E.; Methe´, Barbara; Wu, Martin; Heidelberg, John F.; Paulsen, Ian T.; Fouts, Derrick; Ravel, Jacques; Tettelin, Herve; Ren, Qinghu; Read, Tim; DeBoy, Robert T.; Seshadri, Rekha; Salzberg, Steven L.; Jensen, Harold B.; Birkeland, Nils Kare; Nelson, William C.; Dodson, Robert J.; Grindhaug, Svenn H.; Holt, Ingeborg; Eidhammer, Ingvar; Jonasen, Inge; Vanaken, Susan; Utterback, Terry; Feldblyum, Tamara V.; Fraser, Claire M.; Lillehaug, Johan R.; Eisen, Jonathan A.Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.Item Hawkeye: an interactive visual analytics tool for genome assemblies(Genome Biology, 2007-03-09) Schatz, Michael C.; Phillippy, Adam M.; Shneiderman, Ben; Salzberg, Steven L.Genome sequencing remains an inexact science, and genome sequences can contain significant errors if they are not carefully examined. Hawkeye is our new visual analytics tool for genome assemblies, designed to aid in identifying and correcting assembly errors. Users can analyze all levels of an assembly along with summary statistics and assembly metrics, and are guided by a ranking component towards likely mis-assemblies. Hawkeye is freely available and released as part of the open source AMOS project http://amos.sourceforge.net/hawkeye.Item Identifying bacterial genes and endosymbiont DNA with Glimmer(Bioinformatics, 2007-03) Delcher, Arthur L.; Bratke, Kirsten A.; Powers, Edwin C.; Salzberg, Steven L.Motivation: The Glimmer gene-finding software has been successfully used for finding genes in bacteria, archæa and viruses representing hundreds of species. We describe several major changes to the Glimmer system, including improved methods for identifying both coding regions and start codons. We also describe a new module of Glimmer that can distinguish host and endosymbiont DNA. This module was developed in response to the discovery that eukaryotic genome sequencing projects sometimes inadvertently capture the DNA of intracellular bacteria living in the host. Results: The new methods dramatically reduce the rate of falsepositive predictions, while maintaining Glimmer’s 99% sensitivity rate at detecting genes in most species, and they find substantially more correct start sites, as measured by comparisons to known and well-curated genes. We show that our interpolated Markov model (IMM) DNA discriminator correctly separated 99% of the sequences in a recent genome project that produced a mixture of sequences from the bacterium Prochloron didemni and its sea squirt host, Lissoclinum patella.Item JIGSAW, GeneZilla, and GlimmerHMM: puzzling out the features of human genes in the ENCODE regions(Genome Biology, 2006-08-07) Allen, Jonathan E.; Majoros, William H.; Pertea, Mihaela; Salzberg, Steven L.Background: Predicting complete protein-coding genes in human DNA remains a significant challenge. Though a number of promising approaches have been investigated, an ideal suite of tools has yet to emerge that can provide near perfect levels of sensitivity and specificity at the level of whole genes. As an incremental step in this direction, it is hoped that controlled gene finding experiments in the ENCODE regions will provide a more accurate view of the relative benefits of different strategies for modeling and predicting gene structures. Results: Here we describe our general-purpose eukaryotic gene finding pipeline and its major components, as well as the methodological adaptations that we found necessary in accommodating human DNA in our pipeline, noting that a similar level of effort may be necessary by ourselves and others with similar pipelines whenever a new class of genomes is presented to the community for analysis. We also describe a number of controlled experiments involving the differential inclusion of various types of evidence and feature states into our models and the resulting impact these variations have had on predictive accuracy. Conclusions: While in the case of the non-comparative gene finders we found that adding model states to represent specific biological features did little to enhance predictive accuracy, for our evidence-based ‘combiner’ program the incorporation of additional evidence tracks tended to produce significant gains in accuracy for most evidence types, suggesting that improved modeling efforts at the hidden Markov model level are of relatively little value. We relate these findings to our current plans for future research.Item Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote(PLoS Biology, 2006) Eisen, Jonathan A.; Coyne, Robert S.; Wu, Martin; Wu, Dongying; Thiagarajan, Mathangi; Wortman, Jennifer R.; Badger, Jonathan H.; Ren, Qinghu; Amedeo, Paolo; Jones, Kristie M.; Tallon, Luke J.; Delcher, Arthur L.; Salzberg, Steven L.; Silva, Joana C.; Haas, Brian J.; Majoros, William H.; Farzad, Maryam; Carlton, Jane M.; Smith, Robert K. Jr.; Garg, Jyoti; Pearlman, Ronald E.; Karrer, Kathleen M.; Sun, Lei; Manning, Gerard; Elde, Nels C.; Turkewitz, Aaron P.; Asai, David J.; Wilkes, David E.; Wang, Yufeng; Cai, Hong; Collins, Kathleen; Stewart, B. Andrew; Lee, Suzanne R.; Wilamowsk, Katarzyna; Weinberg, Zasha; Ruzzo, Walter L.; Wloga, Dorota; Gaertig, Jacek; Frankel, Joseph; Tsao, Che-Chia; Gorovsky, Martin A.; Keeling, Patrick J.; Waller, Ross F.; Patron, Nicola J.; Cherry, J. Michael; Stover, Nicholas A.; Krieger, Cynthia J.; del Toro, Christina; Ryder, Hilary F.; Williamson, Sondra C.; Barbeau, Rebecca A.; Hamilton, Eileen P.; Orias, EduardoThe ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.Item Method for Identifying Splice Sites and Translational Start Sites in Eukaryotic mRNA(Computer Applications in the Biosciences (CABIOS), 1997) Salzberg, Steven L.This paper describes a new method for determining the consensus sequences that signal the start of donor translation and the boundaries between exons and introns (donor and acceptor sites) in eukaryotic mRNA. The method takes into account the dependencies between adjacent bases, in contrast to the usual technique of considering each position independently. When coupled with a dynamic program to compute the most likely sequence, new consensus sequences emerge. The consensus sequence information is summarized in conditional probability matrices which, when used to locate signals in uncharacterized genomic DNA, have greater sensitivity and specificity than conventional matrices. Species-specific versions of these matrices are especially effective at distinguishing true and false sites.Item Minimus: a fast, lightweight genome assembler(BMC Bioinformatics, 2007-02-26) Sommer, Daniel D.; Delcher, Arthur L.; Salzberg, Steven L.; Pop, MihaiBackground: Genome assemblers have grown very large and complex in response to the need for algorithms to handle the challenges of large whole-genome sequencing projects. Many of the most common uses of assemblers, however, are best served by a simpler type of assembler that requires fewer software components, uses less memory, and is far easier to install and run. Results: We have developed the Minimus assembler to address these issues, and tested it on a range of assembly problems. We show that Minimus performs well on several small assembly tasks, including the assembly of viral genomes, individual genes, and BAC clones. In addition, we evaluate Minimus' performance in assembling bacterial genomes in order to assess its suitability as a component of a larger assembly pipeline. We show that, unlike other software currently used for these tasks, Minimus produces significantly fewer assembly errors, at the cost of generating a more fragmented assembly. Conclusion: We find that for small genomes and other small assembly tasks, Minimus is faster and far more flexible than existing tools. Due to its small size and modular design Minimus is perfectly suited to be a component of complex assembly pipelines. Minimus is released as an open-source software project and the code is available as part of the AMOS project at Sourceforge.Item Rapid, accurate, computational discovery of Rho-independent transcription terminators illuminates their relationship to DNA uptake(Genome Biology, 2007-02-21) Kingsford, Carleton L.; Ayanbule, Kunmi; Salzberg, Steven L.Background: In many prokaryotes, transcription of DNA to RNA is terminated by a thymine-rich stretch of DNA following a hairpin loop. Detecting such Rho-independent transcription terminators can shed light on the organization of bacterial genomes and can improve genome annotation. Previous computational methods to predict Rho-independent terminators have been slow or limited in the organisms they consider. Results: We describe TransTermHP, a new computational method to rapidly and accurately detect Rho-independent transcription terminators. We predict the locations of terminators in 343 prokaryotic genomes, representing the largest collection of predictions available. In Bacillus subtilis, we can detect 93% of known terminators with a false positive rate of just 6%, comparable to the best-known methods. Outside the Firmicutes division, we find that Rho-independent termination plays a large role in the Neisseria and Vibrio genera, the Pasteurellaceae (including the Haemophilus genus) and several other species. In Neisseria and Pasteurellaceae, terminator hairpins are frequently formed by closely spaced, complementary instances of exogenous DNA uptake signal sequences. We quantify the propensity for terminators to include these sequences. In the process, we provide the first discussion of potential uptake signals in Haemophilus ducreyi and Mannheimia succiniciproducens, and we discuss the preference for a particular configuration of uptake signal sequences within terminators. Conclusion: Our new fast and accurate method for detecting transcription terminators has allowed us to identify and analyze terminators in many new genomes and to identify DNA uptake signal sequences in several species where they have not been previously reported. Our software and predictions are freely available.Item Serendipitous discovery of Wolbachia genomes in multiple Drosophila species(Genome Biology, 2005) Salzberg, Steven L.; Dunning Hotopp, Julie C.; Delcher, Arthur L.; Pop, Mihai; Smith, Douglas R; Eisen, Michael B.; Nelson, William C.Background: The Trace Archive is a repository for the raw, unanalyzed data generated by largescale genome sequencing projects. The existence of this data offers scientists the possibility of discovering additional genomic sequences beyond those originally sequenced. In particular, if the source DNA for a sequencing project came from a species that was colonized by another organism, then the project may yield substantial amounts of genomic DNA, including near-complete genomes, from the symbiotic or parasitic organism. Results: By searching the publicly available repository of DNA sequencing trace data, we discovered three new species of the bacterial endosymbiont Wolbachia pipientis in three different species of fruit fly: Drosophila ananassae, D. simulans, and D. mojavensis. We extracted all sequences with partial matches to a previously sequenced Wolbachia strain and assembled those sequences using customized software. For one of the three new species, the data recovered were sufficient to produce an assembly that covers more than 95% of the genome; for a second species the data produce the equivalent of a 'light shotgun' sampling of the genome, covering an estimated 75-80% of the genome; and for the third species the data cover approximately 6-7% of the genome. Conclusions: The results of this study reveal an unexpected benefit of depositing raw data in a central genome sequence repository: new species can be discovered within this data. The differences between these three new Wolbachia genomes and the previously sequenced strain revealed numerous rearrangements and insertions within each lineage and hundreds of novel genes. The three new genomes, with annotation, have been deposited in GenBank.Item A unified model explaining the offsets of overlapping and near-overlapping prokaryotic genes.(Molecular Biology and Evolution, 2007) Kingsford, Carl; Delcher, Arthur L.; Salzberg, Steven L.Overlapping genes are a common phenomenon. Among sequenced prokaryotes, more than 29% of all annotated genes overlap at least 1 of their 2 flanking genes. We present a unified model for the creation and repair of overlaps among adjacent genes where the 3# ends either overlap or nearly overlap. Our model, derived from a comprehensive analysis of complete prokaryotic genomes in GenBank, explains the nonuniform distribution of the lengths of such overlap regions far more simply than previously proposed models. Specifically, we explain the distribution of overlap lengths based on random extensions of genes to the next occurring downstream stop codon. Our model also provides an explanation for a newly observed (here) pattern in the distribution of the separation distances of closely spaced nonoverlapping genes. We provide evidence that the newly described biased distribution of separation distances is driven by the same phenomenon that creates the uneven distribution of overlap lengths. This suggests a dynamic picture of continual overlap creation and elimination.Item Use and mis-use of supplementary material in science publications(Springer Nature, 2015-11-03) Pop, Mihai; Salzberg, Steven L.Supplementary material is a ubiquitous feature of scientific articles, particularly in journals that limit the length of the articles. While the judicious use of supplementary material can improve the readability of scientific articles, its excessive use threatens the scientific review process and by extension the integrity of the scientific literature. In many cases supplementary material today is so extensive that it is reviewed superficially or not at all. Furthermore, citations buried within supplementary files rob other scientists of recognition of their contribution to the scientific record. These issues are exacerbated by the lack of guidance on the use of supplementary information from the journals to authors and reviewers. We propose that the removal of artificial length restrictions plus the use of interactive features made possible by modern electronic media can help to alleviate these problems. Many journals, in fact, have already removed article length limitations (as is the case for BMC Bioinformatics and other BioMed Central journals). We hope that the issues raised in our article will encourage publishers and scientists to work together towards a better use of supplementary information in scientific publishing.Item Versatile and open software for comparing large genomes(Genome Biology, 2004-01-30) Kurtz, Stefan; Phillippy, Adam; Delcher, Arthur L.; Smoot, Michael; Shumway, Martin; Antonescu, Corina; Salzberg, Steven L.The newest version of MUMmer easily handles comparisons of large eukaryotic genomes at varying evolutionary distances, as demonstrated by applications to multiple genomes. Two new graphical viewing tools provide alternative ways to analyze genome alignments. The new system is the first version of MUMmer to be released as open-source software. This allows other developers to contribute to the code base and freely redistribute the code. The MUMmer sources are available at http://www.tigr.org/software/mummer.Item Whole-Genome Analysis of Human Influenza A Virus Reveals Multiple Persistent Lineages and Reassortment among Recent H3N2 Viruses(PLoS Biology, 2005) Holmes, Edward C.; Ghedin, Elodie; Miller, Naomi; Taylor, Jill; Bao, Yiming; St. George, Kirsten; Grenfell, Bryan T.; Salzberg, Steven L.; Fraser, Claire M.; Lipman, David J.; Taubenberger, Jeffery K.Understanding the evolution of influenza A viruses in humans is important for surveillance and vaccine strain selection. We performed a phylogenetic analysis of 156 complete genomes of human H3N2 influenza A viruses collected between 1999 and 2004 from New York State, United States, and observed multiple co-circulating clades with different population frequencies. Strikingly, phylogenies inferred for individual gene segments revealed that multiple reassortment events had occurred among these clades, such that one clade of H3N2 viruses present at least since 2000 had provided the hemagglutinin gene for all those H3N2 viruses sampled after the 2002–2003 influenza season. This reassortment event was the likely progenitor of the antigenically variant influenza strains that caused the A/Fujian/411/2002-like epidemic of the 2003–2004 influenza season. However, despite sharing the same hemagglutinin, these phylogenetically distinct lineages of viruses continue to co-circulate in the same population. These data, derived from the first large-scale analysis of H3N2 viruses, convincingly demonstrate that multiple lineages can co-circulate, persist, and reassort in epidemiologically significant ways, and underscore the importance of genomic analyses for future influenza surveillance.