De novo assembly using low-coverage short read sequence data from the rice pathogen Pseudomonas syringae pv. oryzae.

We developed a novel approach for de novo genome assembly using only sequence data from high-throughput short read sequencing technologies. By combining data generated from 454 Life Sciences (Roche) and Illumina (formerly known as Solexa sequencing) sequencing platforms, we reliably assembled genomes into large scaffolds at a fraction of the traditional cost and without use of a reference sequence. We applied this method to two isolates of the phytopathogenic bacteria Pseudomonas syringae. Sequencing and reassembly of the well-studied tomato and Arabidopsis pathogen, Pto(DC3000), facilitated development and testing of our method. Sequencing of a distantly related rice pathogen, Por(1_)(6), demonstrated our method's efficacy for de novo assembly of novel genomes. Our assembly of Por(1_6) yielded an N50 scaffold size of 531,821 bp with >75% of the predicted genome covered by scaffolds over 100,000 bp. One of the critical phenotypic differences between strains of P. syringae is the range of plant hosts they infect. This is largely determined by their complement of type III effector proteins. The genome of Por(1_6) is the first sequenced for a P. syringae isolate that is a pathogen of monocots, and, as might be predicted, its complement of type III effectors differs substantially from the previously sequenced isolates of this species. The genome of Por(1_6) helps to define an expansion of the P. syringae pan-genome, a corresponding contraction of the core genome, and a further diversification of the type III effector complement for this important plant pathogen species.

[1]  F. Sanger,et al.  A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. , 1975, Journal of molecular biology.

[2]  A. Cornish-Bowden.  Nomenclature for incompletely specified bases in nucleic acid sequences: recommendations 1984. , 1985, Nucleic acids research.

[3]  C. Bender,et al.  Reduced pathogen fitness of Pseudomonas syringae pv. tomato Tn5 mutants defective in coronatine production , 1987 .

[4]  B. Staskawicz,et al.  Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. , 1989, Science.

[5]  S. Hutcheson,et al.  A single promoter sequence recognized by a newly identified alternate sigma factor directs expression of pathogenicity and host range determinants in Pseudomonas syringae , 1994, Journal of bacteriology.

[6]  S. Lewis,et al.  Genome annotation assessment in Drosophila melanogaster. , 2000, Genome research.

[7]  J. Hacker,et al.  Pathogenicity islands and the evolution of microbes. , 2000, Annual review of microbiology.

[8]  R. Guigó,et al.  An assessment of gene prediction accuracy in large DNA sequences. , 2000, Genome research.

[9]  J. Mansfield,et al.  Excision from tRNA genes of a large chromosomal region, carrying avrPphB, associated with race change in the bean pathogen, Pseudomonas syringae pv. phaseolicola , 2000, Molecular microbiology.

[10]  N. Moran,et al.  Genes Lost and Genes Found: Evolution of Bacterial Pathogenesis and Symbiosis , 2001, Science.

[11]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[12]  J. Dangl,et al.  Nucleotide sequence, functional characterization and evolution of pFKN, a virulence plasmid in Pseudomonas syringae pathovar maculicola , 2003, Molecular microbiology.

[13]  Jia Liu,et al.  The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Ullrich,et al.  Complete nucleotide sequence and analysis of pPSR1 (72,601 bp), a pPT23A-family plasmid from Pseudomonas syringae pv. syringae A2 , 2003, Molecular Genetics and Genomics.

[15]  Mark Borodovsky,et al.  Prokaryotic Gene Prediction Using GeneMark and GeneMark.hmm , 2003, Current protocols in bioinformatics.

[16]  Adam M. Phillippy,et al.  Comparative genome assembly , 2004, Briefings Bioinform..

[17]  Sheng Yang He,et al.  Type III protein secretion mechanism in mammalian and plant pathogens. , 2004, Biochimica et biophysica acta.

[18]  D. Guttman,et al.  Nucleotide Sequence and Evolution of the Five-Plasmid Complement of the Phytopathogen Pseudomonas syringae pv. maculicola ES4326 , 2004, Journal of bacteriology.

[19]  Mark Borodovsky,et al.  GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses , 2005, Nucleic Acids Res..

[20]  David S. Guttman,et al.  Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae , 2005, Applied and Environmental Microbiology.

[21]  Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition. , 2005, Journal of bacteriology.

[22]  J. Mansfield,et al.  Exposure to Host Resistance Mechanisms Drives Evolution of Bacterial Virulence in Plants , 2005, Current Biology.

[23]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[24]  Matt Nolan,et al.  Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. B. Mudgett.  New insights to the function of phytopathogenic bacterial type III effectors in plants. , 2005, Annual review of plant biology.

[26]  Jeff H. Chang,et al.  A high-throughput, near-saturating screen for type III effector genes from Pseudomonas syringae. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Jonathan D. G. Jones,et al.  The plant immune system , 2006, Nature.

[28]  David S Guttman,et al.  Type III Effector Diversification via Both Pathoadaptation and Horizontal Transfer in Response to a Coevolutionary Arms Race , 2006, PLoS genetics.

[29]  S. Chisholm,et al.  Host-Microbe Interactions: Shaping the Evolution of the Plant Immune Response , 2022 .

[30]  A. Halpern,et al.  A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[31]  L. M. Schechter,et al.  Multiple approaches to a complete inventory of Pseudomonas syringae pv. tomato DC3000 type III secretion system effector proteins. , 2006, Molecular plant-microbe interactions : MPMI.

[32]  J. Boch,et al.  Different versions of Pseudomonas syringae pv. tomato DC3000 exist due to the activity of an effector transposon. , 2006, Molecular plant pathology.

[33]  David S Guttman,et al.  Terminal Reassortment Drives the Quantum Evolution of Type III Effectors in Bacterial Pathogens , 2006, PLoS pathogens.

[34]  Jeff H. Chang,et al.  Subterfuge and manipulation: type III effector proteins of phytopathogenic bacteria. , 2006, Annual review of microbiology.

[35]  Justin S. Hogg,et al.  Characterization and modeling of the Haemophilus influenzae core and supragenomes based on the complete genomic sequences of Rd and 12 clinical nontypeable strains , 2007, Genome Biology.

[36]  Vincent J. Magrini,et al.  Extending assembly of short DNA sequences to handle error , 2007, Bioinform..

[37]  N. Moran,et al.  Parallel genomic evolution and metabolic interdependence in an ancient symbiosis , 2007, Proceedings of the National Academy of Sciences.

[38]  Steven Salzberg,et al.  Identifying bacterial genes and endosymbiont DNA with Glimmer , 2007, Bioinform..

[39]  M. Brent.  How does eukaryotic gene prediction work? , 2007, Nature Biotechnology.

[40]  René L. Warren,et al.  Assembling millions of short DNA sequences using SSAKE , 2006, Bioinform..

[41]  Juliane C. Dohm,et al.  SHARCGS, a fast and highly accurate short-read assembly algorithm for de novo genomic sequencing. , 2007, Genome research.

[42]  Ryan D. Morin,et al.  Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. , 2008, Genome research.

[43]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[44]  Mark J. P. Chaisson,et al.  Short read fragment assembly of bacterial genomes. , 2008, Genome research.

[45]  Steven Salzberg,et al.  Gene-Boosted Assembly of a Novel Bacterial Genome from Very Short Reads , 2008, PLoS Comput. Biol..

[46]  David Hernández,et al.  De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. , 2008, Genome research.

[47]  Anthony L. Schilmiller,et al.  COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine , 2008, Proceedings of the National Academy of Sciences.

[48]  Richard M. Clark,et al.  Sequencing of natural strains of Arabidopsis thaliana with short reads. , 2008, Genome research.

[49]  Gabor T. Marth,et al.  Whole-genome sequencing and variant discovery in C. elegans , 2008, Nature Methods.

[50]  Ruiqiang Li,et al.  SOAP: short oligonucleotide alignment program , 2008, Bioinform..

[51]  Michael Q. Zhang,et al.  Scanning the human genome at kilobase resolution. , 2008, Genome research.

[52]  John Stavrinides,et al.  Host–pathogen interplay and the evolution of bacterial effectors , 2007, Cellular microbiology.

[53]  E. Mardis.  The impact of next-generation sequencing technology on genetics. , 2008, Trends in genetics : TIG.

[54]  S. Salzberg,et al.  Bioinformatics challenges of new sequencing technology. , 2008, Trends in genetics : TIG.

[55]  C. Myers,et al.  Roadmap to new virulence determinants in Pseudomonas syringae: insights from comparative genomics and genome organization. , 2008, Molecular plant-microbe interactions : MPMI.

[56]  J. Setubal,et al.  Role of Recombination in the Evolution of the Model Plant Pathogen Pseudomonas syringae pv. tomato DC3000, a Very Atypical Tomato Strain , 2008, Applied and Environmental Microbiology.