Reducing assembly complexity of microbial genomes with single-molecule sequencing

BackgroundThe short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem.ResultsTo measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.ConclusionsAutomated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

[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]  D. Schwartz,et al.  Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping. , 1993, Science.

[3]  Nikos Kyrpides,et al.  Genomes OnLine Database (GOLD 1.0): a monitor of complete and ongoing genome projects world-wide , 1999, Bioinform..

[4]  S. Salzberg,et al.  The Value of Complete Microbial Genome Sequencing (You Get What You Pay For) , 2002, Journal of bacteriology.

[5]  S. Salzberg,et al.  Versatile and open software for comparing large genomes , 2004, Genome Biology.

[6]  Sean D Hooper,et al.  On the nature of gene innovation: duplication patterns in microbial genomes. , 2003, Molecular biology and evolution.

[7]  T. Wetter,et al.  Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs. , 2004, Genome research.

[8]  N. Moran,et al.  Evolutionary Origins of Genomic Repertoires in Bacteria , 2005, PLoS biology.

[9]  Steven Salzberg,et al.  Beware of mis-assembled genomes , 2005, Bioinform..

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

[11]  L. Hirsch.  Competing interests: none declared. , 2006 .

[12]  M. Schatz,et al.  Genome assembly forensics: finding the elusive mis-assembly , 2008, Genome Biology.

[13]  R. Barrangou,et al.  CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes , 2007, Science.

[14]  Nancy F. Hansen,et al.  Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry , 2008, Nature.

[15]  D. Branton,et al.  The potential and challenges of nanopore sequencing , 2008, Nature Biotechnology.

[16]  Sergey Koren,et al.  Aggressive assembly of pyrosequencing reads with mates , 2008, Bioinform..

[17]  Schuyler F. Baldwin,et al.  The Complete Genome Sequence of Escherichia coli DH10B: Insights into the Biology of a Laboratory Workhorse , 2008, Journal of bacteriology.

[18]  Mihai Pop,et al.  Genome assembly reborn: recent computational challenges , 2009, Briefings Bioinform..

[19]  B. Birren,et al.  Genome Project Standards in a New Era of Sequencing , 2009, Science.

[20]  H. Bayley,et al.  Continuous base identification for single-molecule nanopore DNA sequencing. , 2009, Nature nanotechnology.

[21]  M. Touchon,et al.  Genesis, effects and fates of repeats in prokaryotic genomes. , 2009, FEMS microbiology reviews.

[22]  S. Turner,et al.  Real-Time DNA Sequencing from Single Polymerase Molecules , 2009, Science.

[23]  Mihai Pop,et al.  Assembly complexity of prokaryotic genomes using short reads , 2010, BMC Bioinformatics.

[24]  Mihai Pop,et al.  Parametric Complexity of Sequence Assembly: Theory and Applications to Next Generation Sequencing , 2009, J. Comput. Biol..

[25]  S. Turner,et al.  Real-time DNA sequencing from single polymerase molecules. , 2010, Methods in enzymology.

[26]  Matthew Berriman,et al.  Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology , 2010, Bioinform..

[27]  I-Min A. Chen,et al.  The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata , 2007, Nucleic Acids Res..

[28]  S. Koren,et al.  Assembly algorithms for next-generation sequencing data. , 2010, Genomics.

[29]  G. Timp,et al.  Nanopore Sequencing: Electrical Measurements of the Code of Life , 2010, IEEE Transactions on Nanotechnology.

[30]  Tommy L. Wheeler,et al.  Diversity of Multidrug-Resistant Salmonella enterica Strains Associated with Cattle at Harvest in the United States , 2011, Applied and Environmental Microbiology.

[31]  James H. Bullard,et al.  The origin of the Haitian cholera outbreak strain. , 2011, The New England journal of medicine.

[32]  James H. Bullard,et al.  Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. , 2011, The New England journal of medicine.

[33]  Adam M. Phillippy,et al.  Interactive metagenomic visualization in a Web browser , 2011, BMC Bioinformatics.

[34]  Michael C. Schatz,et al.  Genomic dark matter: the reliability of short read mapping illustrated by the genome mappability score , 2012, Bioinform..

[35]  Tyson A. Clark,et al.  Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing , 2012, Nature Biotechnology.

[36]  Bud Mishra,et al.  Reevaluating Assembly Evaluations with Feature Response Curves: GAGE and Assemblathons , 2012, PloS one.

[37]  Sergey I. Nikolenko,et al.  SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing , 2012, J. Comput. Biol..

[38]  M. Schatz,et al.  Algorithms Gage: a Critical Evaluation of Genome Assemblies and Assembly Material Supplemental , 2008 .

[39]  Gabor T. Marth,et al.  Haplotype-based variant detection from short-read sequencing , 2012, 1207.3907.

[40]  Thomas E. Besser,et al.  Phylogeny of Shiga Toxin-Producing Escherichia coli O157 Isolated from Cattle and Clinically Ill Humans , 2012, Molecular biology and evolution.

[41]  James H. Bullard,et al.  A hybrid approach for the automated finishing of bacterial genomes , 2012, Nature Biotechnology.

[42]  C. Nusbaum,et al.  Finished bacterial genomes from shotgun sequence data , 2012, Genome research.

[43]  I-Min A. Chen,et al.  The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata , 2011, Nucleic Acids Res..

[44]  R. Gibbs,et al.  Mind the Gap: Upgrading Genomes with Pacific Biosciences RS Long-Read Sequencing Technology , 2012, PloS one.

[45]  M. Schatz,et al.  Hybrid error correction and de novo assembly of single-molecule sequencing reads , 2012, Nature Biotechnology.

[46]  Glenn Tesler,et al.  Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory , 2012, BMC Bioinformatics.

[47]  T. Dallman,et al.  Performance comparison of benchtop high-throughput sequencing platforms , 2012, Nature Biotechnology.

[48]  Zhong Wang,et al.  ALE: a generic assembly likelihood evaluation framework for assessing the accuracy of genome and metagenome assemblies , 2013, Bioinform..

[49]  Michael Roberts,et al.  The MaSuRCA genome assembler , 2013, Bioinform..

[50]  Kiyoshi Asai,et al.  PBSIM: PacBio reads simulator - toward accurate genome assembly , 2013, Bioinform..

[51]  M. Pop,et al.  Sequence assembly demystified , 2013, Nature Reviews Genetics.

[52]  Production of Long (1.5kb – 15.0kb), Accurate, DNA Sequencing Reads using an Illumina HiSeq2000 to Support de novo Assembly of the Blue Catfish Genome , 2013 .

[53]  Daniel D. Sommer,et al.  De novo likelihood-based measures for comparing genome assemblies , 2013, BMC Research Notes.

[54]  Aaron A. Klammer,et al.  Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data , 2013, Nature Methods.

[55]  Steven Salzberg,et al.  GAGE-B: an evaluation of genome assemblers for bacterial organisms , 2013, Bioinform..