Ariadna E Morales, Yue Dong, Thomas Brown, Kaushal Baid, Dimitrios-Georgios Kontopoulos, Victoria Gonzalez, Zixia Huang, Alexis-Walid Ahmed, Arkadeb Bhuinya, Leon Hilgers, Sylke Winkler, Graham M Hughes, Xiaomeng Li, Ping Lu, Yixin Yang, Bogdan Kirilenko, Paolo Devanna, Tanya M Lama, Yomiran Nissan, Martin Pippel, Liliana M Dávalos, Sonja Vernes, Sébastien J Puechmaille, Stephen J Rossiter, Yossi Yovel, Joseph B Prescott, Andreas Kurth, David A Ray, Burton K Lim, Eugene W Myers, Emma Teeling, Arinjay Banerjee, Aaron T Irving#, Michael Hiller# Bat genomes illuminate adaptations to viral tolerance and disease resistance. Nature, 638(8050) 449-458 (2025)
Open Access DOI
Zoonoses are infectious diseases transmitted from animals to humans. Bats have been suggested to harbour more zoonotic viruses than any other mammalian order1. Infections in bats are largely asymptomatic2,3, indicating limited tissue-damaging inflammation and immunopathology. To investigate the genomic basis of disease resistance, the Bat1K project generated reference-quality genomes of ten bat species, including potential viral reservoirs. Here we describe a systematic analysis covering 115 mammalian genomes that revealed that signatures of selection in immune genes are more prevalent in bats than in other mammalian orders. We found an excess of immune gene adaptations in the ancestral chiropteran branch and in many descending bat lineages, highlighting viral entry and detection factors, and regulators of antiviral and inflammatory responses. ISG15, which is an antiviral gene contributing to hyperinflammation during COVID-19 (refs. 4,5), exhibits key residue changes in rhinolophid and hipposiderid bats. Cellular infection experiments show species-specific antiviral differences and an essential role of protein conjugation in antiviral function of bat ISG15, separate from its role in secretion and inflammation in humans. Furthermore, in contrast to humans, ISG15 in most rhinolophid and hipposiderid bats has strong anti-SARS-CoV-2 activity. Our work reveals molecular mechanisms that contribute to viral tolerance and disease resistance in bats.
2023
Blair P Bentley#, Tomás Carrasco-Valenzuela, Elisa K S Ramos, Harvinder Pawar, Larissa Souza Arantes, Alana Alexander, Shreya M Banerjee, Patrick Masterson, Martin Kuhlwilm, Martin Pippel, Jacquelyn Mountcastle, Bettina Haase, Marcela Uliano-Silva, Giulio Formenti, Kerstin Howe, William Chow, Alan Tracey, Ying Sims, Sarah Pelan, Jonathan Wood, Kelsey Yetsko, Justin R Perrault, Kelly Stewart, Scott R Benson, Yaniv Levy, Erica V Todd, H Bradley Shaffer, Peter Scott, Brian T Henen, Robert F Murphy, David W Mohr, Alan F Scott, D. Duffy, Neil J Gemmell, Alexander Suh, Sylke Winkler, Francoise Thibaud-Nissen, Mariana F Nery, Tomas Marques-Bonet, Agostinho Antunes, Yaron Tikochinski, Peter H Dutton, Olivier Fedrigo, Eugene W Myers, Erich D Jarvis, Camila J Mazzoni#, Lisa M Komoroske# Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories. Proc Natl Acad Sci U.S.A., 120(7) Art. No. e2201076120 (2023)
Open Access DOI
Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.
2022
Takeshi Takeuchi*, Yoshihiko Suzuki*, Shugo Watabe, Kosuke Nagai, Tetsuji Masaoka, Manabu Fujie, Mayumi Kawamitsu, Noriyuki Satoh, Eugene W Myers A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster. DNA Res, 29(6) Art. No. dsac035 (2022)
Open Access DOI
Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.
Sergey Nurk*, Sergey Koren*, Arang Rhie*, Mikko Rautiainen*, Andrey V Bzikadze, Alla Mikheenko, Mitchell R Vollger, Nicolas Altemose, Lev Uralsky, Ariel Gershman, Sergey Aganezov, Savannah J Hoyt, Mark Diekhans, Glennis A Logsdon, Michael Alonge, Stylianos E Antonarakis, Matthew Borchers, Gerard G Bouffard, Shelise Y Brooks, Gina V Caldas, Nae-Chyun Chen, Haoyu Cheng, Chen-Shan Chin, William Chow, Leonardo G de Lima, Philip C Dishuck, Richard Durbin, Tatiana Dvorkina, Ian T Fiddes, Giulio Formenti, Robert S Fulton, Arkarachai Fungtammasan, Erik Garrison, Patrick G S Grady, Tina A Graves-Lindsay, Ira M Hall, Nancy F Hansen, Gabrielle A Hartley, Marina Haukness, Kerstin Howe, Michael W Hunkapiller, Chirag Jain, Miten Jain, Erich D Jarvis, Peter Kerpedjiev, Melanie Kirsche, Mikhail Kolmogorov, Jonas Korlach, Milinn Kremitzki, Heng Li, Valerie V Maduro, Tobias Marschall, Ann M McCartney, J McDaniel, Danny E Miller, James C Mullikin, Eugene W Myers, Nathan D Olson, Benedict Paten, Paul Peluso, Pavel Pevzner, David Porubsky, Tamara Potapova, Evgeny I Rogaev, Jeffrey A Rosenfeld, Steven L Salzberg, Valerie A Schneider, Fritz J Sedlazeck, Kishwar Shafin, Colin J Shew, Alaina Shumate, Ying Sims, Arian F A Smit, Daniela C Soto, Ivan Sović, Jessica M Storer, Aaron Streets, Beth A Sullivan, Francoise Thibaud-Nissen, James Torrance, Justin Wagner, Brian Walenz, Aaron M Wenger, Jonathan Wood, Chunlin Xiao, Stephanie M Yan, Alice C Young, Samantha Zarate, Urvashi Surti, Rajiv C McCoy, Megan Y Dennis, Ivan A Alexandrov, Jennifer L Gerton, Rachel J O'Neill, Winston Timp, Justin M Zook, Michael C Schatz, Evan E Eichler#, Karen H Miga#, Adam M Phillippy# The complete sequence of a human genome. Science, 376(6588) 44-53 (2022) DOI
Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.
Kang Du, Martin Pippel, Susanne Kneitz, Romain Feron, Irene da Cruz, Sylke Winkler, Brigitta Wilde, Edgar G Avila Luna, Gene Myers, Yann Guiguen, Constantino Macias Garcia, Manfred Schartl Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation. Genome Res, 32(3) 583-594 (2022) DOI
Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.
2021
Yuhan Wang, Mark Eddison, Greg Fleishman, Martin Weigert, Shengjin Xu, Tim Wang, Konrad Rokicki, Cristian Goina, Fredrick E Henry, Andrew L Lemire, Uwe Schmidt, Hui Yang, Karel Svoboda, Eugene W Myers, Stephan Saalfeld, Wyatt Korff, Scott M Sternson#, Paul W Tillberg# EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell, 184(26) 6361-6377 (2021)
Open Access DOI
Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.
Diana D Moreno Santillán, Tanya M Lama, Yocelyn T Gutierrez Guerrero, Alexis M Brown, Paul Donat, Huabin Zhao, Stephen J Rossiter, Laurel R Yohe, Joshua H Potter, Emma Teeling, Sonja Vernes, Kalina T J Davies, Eugene W Myers, Graham M Hughes, Zixia Huang, Federico Hoffmann, Angelique P Corthals, David A Ray#, Liliana M Dávalos# Large-scale genome sampling reveals unique immunity and metabolic adaptations in bats. Mol Ecol, 30(23) 6449-6467 (2021) DOI
Comprising more than 1,400 species, bats possess adaptations unique among mammals including powered flight, unexpected longevity, and extraordinary immunity. Some of the molecular mechanisms underlying these unique adaptations includes DNA repair, metabolism and immunity. However, analyses have been limited to a few divergent lineages, reducing the scope of inferences on gene family evolution across the Order Chiroptera. We conducted an exhaustive comparative genomic study of 37 bat species, one generated in this study, encompassing a large number of lineages, with a particular emphasis on multi-gene family evolution across immune and metabolic genes. In agreement with previous analyses, we found lineage-specific expansions of the APOBEC3 and MHC-I gene families, and loss of the proinflammatory PYHIN gene family. We inferred more than 1,000 gene losses unique to bats, including genes involved in the regulation of inflammasome pathways such as epithelial defence receptors, the natural killer gene complex and the interferon-gamma induced pathway. Gene set enrichment analyses revealed genes lost in bats are involved in defence response against pathogen-associated molecular patterns and damage-associated molecular patterns. Gene family evolution and selection analyses indicate bats have evolved fundamental functional differences compared to other mammals in both innate and adaptive immune system, with the potential to enhance antiviral immune response while dampening inflammatory signalling. In addition, metabolic genes have experienced repeated expansions related to convergent shifts to plant-based diets. Our analyses support the hypothesis that, in tandem with flight, ancestral bats had evolved a unique set of immune adaptations whose functional implications remain to be explored.
Giulio Formenti#, Arang Rhie, Jennifer Balacco, Bettina Haase, Jacquelyn Mountcastle, Olivier Fedrigo, Samara Brown, Marco Rosario Capodiferro, Farooq O Al-Ajli, Roberto Ambrosini, Peter Houde, Sergey Koren, Karen Oliver, Michelle Smith, Jason Skelton, Emma Betteridge, Jale Dolucan, Craig Corton, Iliana Bista, James Torrance, Alan Tracey, Jonathan Wood, Marcela Uliano-Silva, Kerstin Howe, Shane A McCarthy, Sylke Winkler, Woori Kwak, Jonas Korlach, Arkarachai Fungtammasan, Daniel Fordham, Vania Costa, Simon Mayes, Matteo Chiara, David S Horner, Eugene W Myers, Richard Durbin, Alessandro Achilli, Edward L Braun, Adam M Phillippy, Erich D Jarvis#, Erich D null Complete vertebrate mitogenomes reveal widespread repeats and gene duplications. Genome Biol, 22(1) Art. No. 120 (2021)
Open Access DOI
Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly.
Arang Rhie, Shane A McCarthy, Olivier Fedrigo, Joana Damas, Giulio Formenti, Sergey Koren, Marcela Uliano-Silva, William Chow, Arkarachai Fungtammasan, Juwan Kim, Chul Lee, Byung June Ko, Mark Chaisson, Gregory L Gedman, Lindsey J Cantin, Francoise Thibaud-Nissen, Leanne Haggerty, Iliana Bista, Michelle Smith, Bettina Haase, Jacquelyn Mountcastle, Sylke Winkler, Sadye Paez, Jonathon Howard, Sonja Vernes, Tanya M Lama, Frank Grutzner, Wesley C Warren, Christopher N Balakrishnan, Dave Burt, Julia M George, Matthew T Biegler, David Iorns, Andrew Digby, Daryl Eason, Bruce Robertson, Taylor Edwards, Mark Wilkinson, George Turner, Axel Meyer, Andreas F Kautt, Paolo Franchini, H William Detrich, Hannes Svardal, Maximilian Wagner, Gavin J P Naylor, Martin Pippel, Milan Malinsky, Mark Mooney, Maria Simbirsky, Brett T Hannigan, Trevor Pesout, Marlys Houck, Ann Misuraca, Sarah B Kingan, Richard J Hall, Zev Kronenberg, Ivan Sović, Christopher Dunn, Zemin Ning, Alex R Hastie, Joyce Lee, Siddarth Selvaraj, Richard E Green, Nicholas H Putnam, Ivo Gut, Jay Ghurye, Erik Garrison, Ying Sims, Joanna Collins, Sarah Pelan, James Torrance, Alan Tracey, Jonathan Wood, Robel E Dagnew, Dengfeng Guan, Sarah E London, David F Clayton, Claudio V Mello, Samantha R Friedrich, Peter V Lovell, Ekaterina Osipova, Farooq O Al-Ajli, Simona Secomandi, Heebal Kim, Constantina Theofanopoulou, Michael Hiller, Yang Zhou, Robert S Harris, Kateryna D Makova, Paul Medvedev, Jinna Hoffman, Patrick Masterson, Karen Clark, Fergal Martin, Kerstin Howe#, Paul Flicek, Brian Walenz, Woori Kwak, Hiram Clawson, Mark Diekhans, Luis Nassar, Benedict Paten, Robert H S Kraus, Andrew J Crawford, M Thomas P Gilbert, Guojie Zhang, Byrappa Venkatesh, Robert F Murphy, Klaus-Peter Koepfli, Beth Shapiro, Warren E Johnson, Federica Di Palma, Tomas Marques-Bonet, Emma Teeling, Tandy Warnow, Jennifer Marshall Graves, Oliver A Ryder, David Haussler, Stephen J O'Brien, Jonas Korlach, Harris A Lewin, Kerstin Howe#, Eugene W Myers#, Richard Durbin#, Adam M Phillippy#, Erich D Jarvis# Towards complete and error-free genome assemblies of all vertebrate species. Nature, 592(7856) 737-746 (2021)
Open Access DOI
High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.
2020
Andreas F Kautt, Claudius F Kratochwil, Alexander Nater, Gonzalo Machado-Schiaffino, Melisa Olave, Frederico Henning, Julián Torres-Dowdall, Andreas Härer, C Darrin Hulsey, Paolo Franchini, Martin Pippel, Eugene W Myers, Axel Meyer Contrasting signatures of genomic divergence during sympatric speciation. Nature, 588(7836) 106-111 (2020)
Open Access DOI
The transition from 'well-marked varieties' of a single species into 'well-defined species'-especially in the absence of geographic barriers to gene flow (sympatric speciation)-has puzzled evolutionary biologists ever since Darwin1,2. Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process3. Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs4, but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories5. Here, within a young species complex of neotropical cichlid fishes (Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry.
David Jebb*, Zixia Huang*, Martin Pippel*, Graham M Hughes, Ksenia Lavrichenko, Paolo Devanna, Sylke Winkler, Lars S Jermiin, Emilia C Skirmuntt, Aris Katzourakis, Lucy Burkitt-Gray, David A Ray, Kevin F. Sullivan, Juliana G. Roscito, Bogdan Kirilenko, Liliana M Dávalos, Angelique P Corthals, Megan L Power, Gareth Jones, Roger D Ransome, Dina K N Dechmann, Andrea G Locatelli, Sébastien J Puechmaille, Olivier Fedrigo, Erich D Jarvis, Michael Hiller#, Sonja Vernes#, Eugene W Myers#, Emma Teeling# Six reference-quality genomes reveal evolution of bat adaptations. Nature, 583(7817) 578-584 (2020)
Open Access DOI
Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our 'Tool to infer Orthologs from Genome Alignments' (TOGA) software with de novo and homology gene predictions as well as short- and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease1.
2018
Sergej Nowoshilow, Siegfried Schloissnig, Jifeng Fei, Andreas Dahl, Andy W C Pang, Martin Pippel, Sylke Winkler, Alex R Hastie, George Young, Juliana G. Roscito, Francisco Falcon, Dunja Knapp, Sean Powell, Alfredo Cruz, Han Cao, Bianca Habermann, Michael Hiller, Elly M. Tanaka, Eugene W Myers The axolotl genome and the evolution of key tissue formation regulators. Nature, 554(7690) 50-55 (2018)
Open AccessPDF
DOI
Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/-and Pax7-/-mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.
Matthias Kaiser, Florian Jug, Thomas Julou, Siddharth Deshpande, Thomas Pfohl, Olin Silander#, Gene Myers#, Erik van Nimwegen# Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software. Nat Commun, 9(1) Art. No. 212 (2018)
Open Access DOI
Much is still not understood about how gene regulatory interactions control cell fate decisions in single cells, in part due to the difficulty of directly observing gene regulatory processes in vivo. We introduce here a novel integrated setup consisting of a microfluidic chip and accompanying analysis software that enable long-term quantitative tracking of growth and gene expression in single cells. The dual-input Mother Machine (DIMM) chip enables controlled and continuous variation of external conditions, allowing direct observation of gene regulatory responses to changing conditions in single cells. The Mother Machine Analyzer (MoMA) software achieves unprecedented accuracy in segmenting and tracking cells, and streamlines high-throughput curation with a novel leveraged editing procedure. We demonstrate the power of the method by uncovering several novel features of an iconic gene regulatory program: the induction of Escherichia coli's lac operon in response to a switch from glucose to lactose.
2017
Kamran Rizzolo, Jennifer Huen, Ashwani Kumar, Sadhna Phanse, James Vlasblom, Yoshito Kakihara, Hussein A Zeineddine, Zoran Minic, Jamie Snider, Wen Wang, Carles Pons, Thiago V Seraphim, Edgar Boczek, Simon Alberti, Michael Costanzo, Chad L Myers, Igor Stagljar, Charles Boone, Mohan Babu, Walid A Houry Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping. Cell Rep, 20(11) 2735-2748 (2017)
Open Access DOI
A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.
2016
Kaiser Matthias, Florian Jug, Olin Silander, Siddharth Deshpande, Thomas Pfohl, Thomas Julou#, Gene Myers#, Erik van Nimwegen# Tracking single-cell gene regulation in dynamically controlled environments using an integrated microfluidic and computational setup bioRxiv, Art. No. https://doi.org/10.1101/076224 (2016)
Open Access DOI
L Carine Stapel, Benoit Lombardot, Coleman Broaddus, Dagmar Kainmueller, Florian Jug, Eugene W Myers, Nadine Vastenhouw Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos. Development, 143(3) 540-546 (2016)
Open AccessPDF
DOI
Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos.
2015
Hanchuan Peng, Eugene W Myers Constructing 5D developing gene expression patterns without live animal imaging Biomed Eng Lett, 4(4) 338-346 (2015) DOI
2014
Ellie S Heckscher, Fuhui Long, Michael J Layden, Chein-Hui Chuang, Laurina Manning, Jourdain Richart, Joseph C Pearson, Stephen T Crews, Hanchuan Peng, Gene Myers, Chris Q Doe Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience. Development, 141(12) 2524-2532 (2014) PDF
DOI
A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.
2013
Vineeth Surendranath, Mirko Theis, Bianca Habermann, Frank Buchholz Designing efficient and specific endoribonuclease-prepared siRNAs. Methods Mol Biol, 942 193-204 (2013) PDF
DOI
RNA interference (RNAi) has grown to be one of the main techniques for loss-of-function studies, leading to the elucidation of biological function of genes in various cellular systems and model organisms. While for many invertebrates such as Drosophila melanogaster (D. melanogaster) and Caenorhabditis elegans (C. elegans) long double-stranded RNA (dsRNA) can directly be used to induce a RNAi response, chemically synthesized small interfering RNAs (siRNAs) are typically employed in mammalian cells to avoid an interferon-like response triggered by long dsRNA (Reynolds et al., RNA 12:988-993, 2006). However, siRNAs are expensive and beset with unintentional gene targeting effects (off-targets) confounding the analysis of results from such studies. We, and others, have developed an alternative technology for RNAi in mammalian cells, termed endoribonuclease-prepared siRNA (esiRNA), which is based on the enzymatic generation of siRNA pools by digestion of long dsRNAs with recombinant RNase III in vitro (Yang et al., Proc Natl Acad Sci USA 99: 9942-9947, 2002; Myers et al., Nat Biotechnol 21:324-328; 2003). This technology has proven to be cost-efficient and reliable. Furthermore, several studies have demonstrated that complex pools of siRNAs, as inherent in esiRNAs, which target one transcript reduce off-target effects (Myers et al., J RNAi Gene Silencing 2:181, 2006; Kittler et al., Nat Methods 4:337-344, 2007). Within this chapter we describe design criteria for the generation of target-optimized esiRNAs.
2011
Lei Qu, Fuhui Long, Xiao Liu, Stuart Kim, Eugene Myers, Hanchuan Peng Simultaneous recognition and segmentation of cells: application in C.elegans. Bioinformatics, 27(20) 2895-2902 (2011) PDF
DOI
Automatic recognition of cell identities is critical for quantitative measurement, targeting and manipulation of cells of model animals at single-cell resolution. It has been shown to be a powerful tool for studying gene expression and regulation, cell lineages and cell fates. Existing methods first segment cells, before applying a recognition algorithm in the second step. As a result, the segmentation errors in the first step directly affect and complicate the subsequent cell recognition step. Moreover, in new experimental settings, some of the image features that have been previously relied upon to recognize cells may not be easy to reproduce, due to limitations on the number of color channels available for fluorescent imaging or to the cost of building transgenic animals. An approach that is more accurate and relies on only a single signal channel is clearly desirable.
2009
Xiao Liu, Fuhui Long, Hanchuan Peng, Sarah J Aerni, Min Jiang, Adolfo Sánchez-Blanco, John I Murray, Elicia A. Preston, Barbara Mericle, Serafim Batzoglou, Eugene W Myers, Stuart K Kim Analysis of cell fate from single-cell gene expression profiles in C. elegans. Cell, 139(3) 623-633 (2009) PDF
DOI
The C. elegans cell lineage provides a unique opportunity to look at how cell lineage affects patterns of gene expression. We developed an automatic cell lineage analyzer that converts high-resolution images of worms into a data table showing fluorescence expression with single-cell resolution. We generated expression profiles of 93 genes in 363 specific cells from L1 stage larvae and found that cells with identical fates can be formed by different gene regulatory pathways. Molecular signatures identified repeating cell fate modules within the cell lineage and enabled the generation of a molecular differentiation map that reveals points in the cell lineage when developmental fates of daughter cells begin to diverge. These results demonstrate insights that become possible using computational approaches to analyze quantitative expression from many genes in parallel using a digital gene expression atlas.
Fuhui Long, Hanchuan Peng, Xiao Liu, Stuart K Kim, Eugene Myers A 3D digital atlas of C. elegans and its application to single-cell analyses. Nat Methods, 6(9) 667-672 (2009) PDF
DOI
We built a digital nuclear atlas of the newly hatched, first larval stage (L1) of the wild-type hermaphrodite of Caenorhabditis elegans at single-cell resolution from confocal image stacks of 15 individual worms. The atlas quantifies the stereotypy of nuclear locations and provides other statistics on the spatial patterns of the 357 nuclei that could be faithfully segmented and annotated out of the 558 present at this developmental stage. We then developed an automated approach to assign cell names to each nucleus in a three-dimensional image of an L1 worm. We achieved 86% accuracy in identifying the 357 nuclei automatically. This computational method will allow high-throughput single-cell analyses of the post-embryonic worm, such as gene expression analysis, or ablation or stimulation of cells under computer control in a high-throughput functional screen.
2007
Christopher D Smith, Robert C Edgar, Mark D Yandell, Douglas R Smith, Susan E Celniker, Eugene W Myers, Gary H Karpen Improved repeat identification and masking in Dipterans. Gene, 389(1) 1-9 (2007) PDF
DOI
Repetitive sequences are a major constituent of many eukaryote genomes and play roles in gene regulation, chromosome inheritance, nuclear architecture, and genome stability. The identification of repetitive elements has traditionally relied on in-depth, manual curation and computational determination of close relatives based on DNA identity. However, the rapid divergence of repetitive sequence has made identification of repeats by DNA identity difficult even in closely related species. Hence, the presence of unidentified repeats in genome sequences affects the quality of gene annotations and annotation-dependent analyses (e.g. microarray analyses). We have developed an enhanced repeat identification pipeline using two approaches. First, the de novo repeat finding program PILER-DF was used to identify interspersed repetitive elements in several recently finished Dipteran genomes. Repeats were classified, when possible, according to their similarity to known elements described in Repbase and GenBank, and also screened against annotated genes as one means of eliminating false positives. Second, we used a new program called RepeatRunner, which integrates results from both RepeatMasker nucleotide searches and protein searches using BLASTX. Using RepeatRunner with PILER-DF predictions, we masked repeats in thirteen Dipteran genomes and conclude that combining PILER-DF and RepeatRunner greatly enhances repeat identification in both well-characterized and un-annotated genomes.
Hanchuan Peng, Fuhui Long, Jie Zhou, Garmay Leung, Michael B Eisen, Eugene W Myers Automatic image analysis for gene expression patterns of fly embryos. BMC Cell Biol, 8 Suppl 1 7-7 (2007)
Open AccessPDF
DOI
Staining the mRNA of a gene via in situ hybridization (ISH) during the development of a D. melanogaster embryo delivers the detailed spatio-temporal pattern of expression of the gene. Many biological problems such as the detection of co-expressed genes, co-regulated genes, and transcription factor binding motifs rely heavily on the analyses of these image patterns. The increasing availability of ISH image data motivates the development of automated computational approaches to the analysis of gene expression patterns.
2006
Hanchuan Peng, Fuhui Long, Michael B Eisen, Eugene W Myers Clustering gene expression patterns of fly embryos
In: Proceedings in 3rd IEEE International Symposium on Biomedical Imaging: Macro to Nano
(2006), Piscataway, N.J., IEEE (2006), 1144-1147
PDF
2005
Roded Sharan, Eugene W Myers A motif-based framework for recognizing sequence families. Bioinformatics, 21 Suppl 1 387-393 (2005) PDF
DOI
Many signals in biological sequences are based on the presence or absence of base signals and their spatial combinations. One of the best known examples of this is the signal identifying a core promoter--the site at which the basal transcription machinery starts the transcription of a gene. Our goal is a fully automatic pattern recognition system for a family of sequences, which simultaneously discovers the base signals, their spatial relationships and a classifier based upon them.
2003
A Bernardo Carvalho, Maria D Vibranovski, Joseph W Carlson, Susan E Celniker, Roger A Hoskins, Gerald M Rubin, Granger G Sutton, Mark D Adams, Eugene W Myers, Andrew Clark Y chromosome and other heterochromatic sequences of the Drosophila melanogaster genome: how far can we go? Genetica, 117(2-3) 227-237 (2003) PDF
Whole genome shotgun assemblies have proven remarkably successful in reconstructing the bulk of euchromatic genes, with the only limit appearing to be determined by the sequencing depth. For genes imbedded in heterochromatin, however, the low cloning efficiency of repetitive sequences, combined with the computational challenges, demand that additional clues be used to annotate the sequences. One approach that has proven very successful in identifying protein coding genes in Y-linked heterochromatin of Drosophila melanogaster has been to make a BLASTable database of the small, unmapped contigs and fragments leftover at the end of a shotgun assembly, and to attempt to capture these by blasting with an appropriate query sequence. This approach often yields a staggered alignment of contigs from the unmapped set to the query sequence, as though the disjoint contigs represent small portions of the gene. Further inspection frequently shows that the contigs are broken by very large, heterochromatic introns. Methods of this sort are being expanded to make best use of all available clues to determine which unmapped contigs are associated with genes. These include use of EST libraries, and, in the case of the Y chromosome, testing of male specific genes and reduced shotgun depth of relevant contigs. It appears much more hopeful than anyone would have imagined that whole genome shotgun assemblies can recover the great bulk of even heterochromatic genes.
2002
Richard J Mural, Mark D Adams, Eugene W Myers, Hamilton O Smith, George L Gabor Miklos, Ron Wides, Aaron L. Halpern, Peter W Li, Granger G Sutton, Joe Nadeau, Steven L Salzberg, Robert A Holt, Chinnappa Kodira, Fu Lu, Lin Chen, Zuoming Deng, Carlos C Evangelista, Weiniu Gan, Thomas J Heiman, Jiayin Li, Zhenya Li, Gennady V Merkulov, Natalia V Milshina, Ashwinikumar K Naik, Rong Qi, Bixiong Chris Shue, Aihui Wang, Jian Wang, Xin Wang, Xianghe Yan, Jane Ye, Shibu Yooseph, Qi Zhao, Liansheng Zheng, Shiaoping C Zhu, Kendra Biddick, R A Bolanos, A L Delcher, I M Dew, D P Fasulo, M J Flanigan, Daniel H. Huson, S A Kravitz, Jason R Miller, C M Mobarry, Knut Reinert, K A Remington, Qinyu Zhang, Xiangqun H Zheng, D R Nusskern, Zhongwu Lai, Yiding Lei, Wenyan Zhong, Alison Yao, Ping Guan, Rui-Ru Ji, Zhiping Gu, Zhen-Yuan Wang, Fei Zhong, Chunlin Xiao, Chia-Chien Chiang, Mark D Yandell, Jennifer R Wortman, Peter G Amanatides, Susanne L Hladun, Eric C Pratts, Jeffery E Johnson, Kristina L Dodson, Kerry J Woodford, Cheryl A Evans, Barry Gropman, Douglas B Rusch, Eli Venter, Mei Wang, Thomas J Smith, Jarrett T Houck, Donald E Tompkins, Charles Haynes, Debbie Jacob, Soo H Chin, David R Allen, Carl E Dahlke, Robert Sanders, Kelvin Li, Xiangjun Liu, Alex Levitsky, William H Majoros, Quan Chen, Ashley C Xia, John R Lopez, Michael T Donnelly, Matthew H Newman, Anna Glodek, Cheryl L Kraft, Marc Nodell, Feroze Ali, Hui-Jin An, Danita Baldwin-Pitts, Karen Y Beeson, Shuang Cai, Mark Carnes, Amy Carver, Parris M Caulk, Angela Center, Yen-Hui Chen, Ming-Lai Cheng, My D Coyne, Michelle Crowder, Steven Danaher, Lionel B Davenport, Raymond Desilets, Susanne M Dietz, Lisa Doup, Patrick Dullaghan, Steven Ferriera, Carl R Fosler, Harold C Gire, Andres Gluecksmann, Jeannine D Gocayne, Jonathan Gray, Brit Hart, Jason Haynes, Jeffrey Hoover, Tim Howland, Chinyere Ibegwam, Mena Jalali, David Johns, Leslie Kline, Daniel S Ma, Steven MacCawley, Anand Magoon, Felecia Mann, David May, Tina C McIntosh, Somil Mehta, Linda Moy, Mee C Moy, Brian J Murphy, Sean D Murphy, Keith A Nelson, Zubeda Nuri, Kimberly A Parker, Alexandre C Prudhomme, Vinita N Puri, Hina Qureshi, John C Raley, Matthew S Reardon, Megan A Regier, Yu-Hui C Rogers, Deanna L Romblad, Jakob Schutz, John L Scott, Richard Scott, Cynthia D Sitter, Michella Smallwood, Andrew Sprague, Erin Stewart, Renee Strong, Ellen Suh, Karena Sylvester, Reginald Thomas, Ni Ni Tint, Christopher Tsonis, Gary Wang, George Wang, Monica S Williams, Sherita M Williams, Sandra M Windsor, Keriellen Wolfe, Mitchell M Wu, Jayshree Zaveri, Kabir Chaturvedi, Andrei E Gabrielian, Zhaoxi Ke, Jingtao Sun, Gangadharan Subramanian, J Craig Venter, Cynthia Pfannkoch, Mary Barnstead, Lisa D Stephenson A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome. Science, 296(5573) 1661-1671 (2002) PDF
DOI
The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.
2001
J C Venter, M D Adams, E W Myers, P W Li, Richard J Mural, Granger G Sutton, H O Smith, Mark D Yandell, Cheryl A Evans, Robert A Holt, Jeannine D Gocayne, Peter G Amanatides, R M Ballew, Daniel H. Huson, Jennifer R Wortman, Qinyu Zhang, Chinnappa Kodira, X H Zheng, L Chen, M Skupski, G Subramanian, P D Thomas, J Zhang, George L Gabor Miklos, C Nelson, Samuel Broder, Andrew Clark, Joe Nadeau, V A McKusick, N Zinder, A J Levine, R J Roberts, M Simon, C Slayman, Michael W Hunkapiller, R A Bolanos, A L Delcher, I M Dew, D P Fasulo, M J Flanigan, Liliana Florea, Aaron L. Halpern, Sridhar Hannenhalli, S A Kravitz, S Levy, C M Mobarry, Knut Reinert, K A Remington, J Abu-Threideh, E M Beasley, Kendra Biddick, V Bonazzi, R C Brandon, M Cargill, I Chandramouliswaran, Rosane Charlab, Kabir Chaturvedi, Zuoming Deng, V Di Francesco, P Dunn, K Eilbeck, Carlos C Evangelista, Andrei E Gabrielian, Weiniu Gan, W Ge, F Gong, Z Gu, Ping Guan, T J Heiman, M E Higgins, Rui-Ru Ji, Z Ke, K A Ketchum, Z Lai, Y Lei, Z Li, J Li, Y Liang, X Lin, F Lu, Gennady V Merkulov, Natalia V Milshina, H M Moore, Ashwinikumar K Naik, V A Narayan, B Neelam, D R Nusskern, Douglas B Rusch, Steven L Salzberg, W Shao, Bixiong Chris Shue, J Sun, Z Wang, A Wang, X Wang, J Wang, M Wei, Ron Wides, C Xiao, C Yan, Alison Yao, Jane Ye, M Zhan, W Zhang, H Zhang, Q Zhao, L Zheng, F Zhong, W Zhong, S Zhu, Senming Zhao, D Gilbert, S Baumhueter, G Spier, Crystal N. Carter, Anibal Cravchik, T Woodage, F Ali, Hui-Jin An, A Awe, Danita Baldwin, Holly Baden, Mary Barnstead, I Barrow, Karen Y Beeson, D Busam, Amy Carver, Angela Center, M L Cheng, L Curry, Steven Danaher, Lionel B Davenport, Raymond Desilets, Susanne M Dietz, Kristina L Dodson, Lisa Doup, Steven Ferriera, N Garg, Andres Gluecksmann, Brit Hart, J Haynes, C Haynes, C Heiner, Susanne L Hladun, D Hostin, Jarrett T Houck, Tim Howland, Chinyere Ibegwam, J Johnson, Francis Kalush, Leslie Kline, S Koduru, A Love, F Mann, David May, S McCawley, T McIntosh, I McMullen, M Moy, L Moy, B Murphy, K Nelson, Cynthia Pfannkoch, Eric C Pratts, V Puri, Hina Qureshi, Matthew S Reardon, R Rodriguez, Y H Rogers, Deanna L Romblad, B Ruhfel, R Scott, Cynthia D Sitter, Michella Smallwood, E Stewart, Renee Strong, E Suh, R Thomas, Ni Ni Tint, S Tse, C Vech, G Wang, J Wetter, S Williams, M Williams, Sandra M Windsor, E Winn-Deen, Keriellen Wolfe, Jayshree Zaveri, K Zaveri, Josep F Abril, R Guigó, M J Campbell, K V Sjolander, B Karlak, A Kejariwal, H Mi, B Lazareva, T Hatton, A Narechania, K Diemer, A Muruganujan, N Guo, S Sato, V Bafna, Sorin Istrail, Ross Lippert, R Schwartz, Brian Walenz, Shibu Yooseph, D Allen, A Basu, J Baxendale, L Blick, M Caminha, J Carnes-Stine, Parris M Caulk, Y H Chiang, My D Coyne, Carl E Dahlke, A Mays, M Dombroski, Michael T Donnelly, D Ely, S Esparham, Carl R Fosler, Harold C Gire, S Glanowski, K Glasser, Anna Glodek, M Gorokhov, K Graham, Barry Gropman, M Harris, J Heil, S Henderson, Jeffrey Hoover, D Jennings, C M Jordan, J Jordan, J Kasha, L Kagan, C Kraft, Alex Levitsky, M Lewis, X Liu, J Lopez, D Ma, William H Majoros, J McDaniel, S Murphy, Matthew H Newman, T Nguyen, N Nguyen, Marc Nodell, S Pan, J Peck, M Peterson, W Rowe, Robert Sanders, J Scott, M Simpson, T Smith, Andrew Sprague, T Stockwell, R Turner, E Venter, M Wang, M Wen, D Wu, M Wu, Ashley C Xia, A Zandieh, X Zhu The sequence of the human genome. Science, 291(5507) 1304-1351 (2001) PDF
DOI
A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
2000
M D Adams, Susan E Celniker, Robert A Holt, Cheryl A Evans, Jeannine D Gocayne, Peter G Amanatides, Steven E Scherer, P W Li, Roger A Hoskins, R F Galle, Reed A George, S E Lewis, Stephen Richards, Michael Ashburner, S Henderson, Granger G Sutton, Jennifer R Wortman, Mark D Yandell, Qinyu Zhang, L X Chen, R C Brandon, Y H Rogers, R G Blazej, Mark Champe, B D Pfeiffer, Kenneth Wan, C Doyle, E G Baxter, G Helt, C R Nelson, G L Gabor, Josep F Abril, A Agbayani, Hui-Jin An, C Andrews-Pfannkoch, Danita Baldwin, R M Ballew, A Basu, J Baxendale, L Bayraktaroglu, E M Beasley, Karen Y Beeson, P V Benos, Benjamin P Berman, D Bhandari, S Bolshakov, D Borkova, M R Botchan, J Bouck, P Brokstein, P Brottier, K C Burtis, D Busam, H Butler, E Cadieu, Angela Center, I Chandra, J M Cherry, S Cawley, Carl E Dahlke, Lionel B Davenport, P Davies, B de Pablos, A L Delcher, Zuoming Deng, A Mays, I M Dew, Susanne M Dietz, Kristina L Dodson, Lisa Doup, M Downes, S Dugan-Rocha, B C Dunkov, P Dunn, K J Durbin, Carlos C Evangelista, C Ferraz, Steven Ferriera, W Fleischmann, Carl R Fosler, Andrei E Gabrielian, N Garg, W M Gelbart, K Glasser, Anna Glodek, F Gong, J H Gorrell, Z Gu, Ping Guan, M Harris, N L Harris, D Harvey, T J Heiman, J R Hernandez, Jarrett T Houck, D Hostin, K A Houston, Tim Howland, M H Wei, Chinyere Ibegwam, Mena Jalali, Francis Kalush, Gary H Karpen, Z Ke, J A Kennison, K A Ketchum, B E Kimmel, Chinnappa Kodira, C Kraft, S A Kravitz, D Kulp, Z Lai, P Lasko, Y Lei, Alex Levitsky, J Li, Z Li, Y Liang, X Lin, X Liu, B Mattei, T C McIntosh, M P McLeod, D McPherson, Gennady V Merkulov, Natalia V Milshina, C M Mobarry, J Morris, A Moshrefi, S M Mount, M Moy, B Murphy, L Murphy, Donna M Muzny, D L Nelson, D R Nelson, K A Nelson, K Nixon, D R Nusskern, Joanne M Pacleb, M Palazzolo, G S Pittman, S Pan, J Pollard, V Puri, M G Reese, Knut Reinert, K A Remington, R D Saunders, F Scheeler, H Shen, Bixiong Chris Shue, I Sidén-Kiamos, M Simpson, M Skupski, T Smith, E Spier, A C Spradling, Mark Stapleton, Renee Strong, E Sun, Robert R Svirskas, C Tector, R Turner, E Venter, A H Wang, X Wang, Z Y Wang, D A Wassarman, George Weinstock, Jean Weissenbach, S M Williams, S M WoodageT, K C Worley, D Wu, S Yang, Q A Yao, Jane Ye, R F Yeh, J S Zaveri, M Zhan, G Zhang, Q Zhao, L Zheng, X H Zheng, F N Zhong, W Zhong, X Zhou, S Zhu, X Zhu, H O Smith, Richard A Gibbs, E W Myers, G M Rubin, J C Venter The genome sequence of Drosophila melanogaster. Science, 287(5461) 2185-2195 (2000) PDF
The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.
1996
Eugene W Myers, Sanford Selznick#, Zheng Zhang#, Webb Miller# Progressive Multiple Alignment with Constraints J Comput Biol, 3(4) 563-572 (1996) PDF
A progressive alignment algorithm produces a multi-alignment of a set of sequences by repeatedly aligning pairs of sequences and/or previously gen- erated alignments. We describe a method for guaranteeing that the alignment generated by a progressive alignment strategy satisfies a user-specified collec- tion of constraints about where certain sequence positions should appear rel- ative to others. Given a collection of constraints over sequences whose
total length is , our algorithm takes time. An alignment of the -like globin gene clusters of several mammals illustrates the practi- cality of the method.
1990
S F Altschul, W Gish, W Miller, E W Myers, D J Lipman Basic local alignment search tool. J Mol Biol, 215(3) 403-410 (1990) PDF
DOI
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
