title: Enhanced de novo assembly of high throughput pyrosequencing data using whole genome mapping.
abstract: Despite major advances in next-generation sequencing, assembly of sequencing data, especially data from novel microorganisms or re-emerging pathogens, remains constrained by the lack of suitable reference sequences. De novo assembly is the best approach to achieve an accurate finished sequence, but multiple sequencing platforms or paired-end libraries are often required to achieve full genome coverage. In this study, we demonstrated a method to assemble complete bacterial genome sequences by integrating shotgun Roche 454 pyrosequencing with optical whole genome mapping (WGM). The whole genome restriction map (WGRM) was used as the reference to scaffold de novo assembled sequence contigs through a stepwise process. Large de novo contigs were placed in the correct order and orientation through alignment to the WGRM. De novo contigs that were not aligned to WGRM were merged into scaffolds using contig branching structure information. These extended scaffolds were then aligned to the WGRM to identify the overlaps to be eliminated and the gaps and mismatches to be resolved with unused contigs. The process was repeated until a sequence with full coverage and alignment with the whole genome map was achieved. Using this method we were able to achieved 100% WGRM coverage without a paired-end library. We assembled complete sequences for three distinct genetic components of a clinical isolate of Providencia stuartii: a bacterial chromosome, a novel bla NDM-1 plasmid, and a novel bacteriophage, without separately purifying them to homogeneity.
authors.affiliation: Multidrug-Resistant Organism Surveillance Network and Repository, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America. Fatma.Onmus-Leone.ctr@us.army.mil
authors.forename: Fatma
authors.initials: F
authors.lastname: Onmus-Leone
authors.forename: Jun
authors.initials: J
authors.lastname: Hang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Clifford
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Riley
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kuschner
authors.forename: Paige E
authors.initials: PE
authors.lastname: Waterman
authors.forename: Emil P
authors.initials: EP
authors.lastname: Lesho
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D011532:Providencia / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D012335:RNA, Ribosomal