Dataset Information

Phased diploid genome assembly with single-molecule real-time sequencing.

ABSTRACT: While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.

SUBMITTER: Chin CS

PROVIDER: S-EPMC5503144 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Phased diploid genome assembly with single-molecule real-time sequencing.

Chin Chen-Shan CS Peluso Paul P Sedlazeck Fritz J FJ Nattestad Maria M Concepcion Gregory T GT Clum Alicia A Dunn Christopher C O'Malley Ronan R Figueroa-Balderas Rosa R Morales-Cruz Abraham A Cramer Grant R GR Delledonne Massimo M Luo Chongyuan C Ecker Joseph R JR Cantu Dario D Rank David R DR Schatz Michael C MC

Nature methods 20161017 12

While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F ...[more]

PMID: 27749838

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Project description:Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged, reducing treatment options for these infections. MDR E. faecalis strains have large genomes containing mobile genetic elements (MGEs) that harbor genes for antibiotic resistance and virulence determinants. Bacteria commonly possess genome defense mechanisms to block MGE acquisition, and we hypothesize that these mechanisms have been compromised in MDR E. faecalis. In restriction-modification (R-M) defense, the bacterial genome is methylated at cytosine (C) or adenine (A) residues by a methyltransferase (MTase), such that nonself DNA can be distinguished from self DNA. A cognate restriction endonuclease digests improperly modified nonself DNA. Little is known about R-M in E. faecalis. Here, we use genome resequencing to identify DNA modifications occurring in the oral isolate OG1RF. OG1RF has one of the smallest E. faecalis genomes sequenced to date and possesses few MGEs. Single-molecule real-time (SMRT) and bisulfite sequencing revealed that OG1RF has global 5-methylcytosine (m5C) methylation at 5'-GCWGC-3' motifs. A type II R-M system confers the m5C modification, and disruption of this system impacts OG1RF electrotransformability and conjugative transfer of an antibiotic resistance plasmid. A second DNA MTase was poorly expressed under laboratory conditions but conferred global N(4)-methylcytosine (m4C) methylation at 5'-CCGG-3' motifs when expressed in Escherichia coli. Based on our results, we conclude that R-M can act as a barrier to MGE acquisition and likely influences antibiotic resistance gene dissemination in the E. faecalis species.The horizontal transfer of antibiotic resistance genes among bacteria is a critical public health concern. Enterococcus faecalis is an opportunistic pathogen that causes life-threatening infections in humans. Multidrug resistance acquired by horizontal gene transfer limits treatment options for these infections. In this study, we used innovative DNA sequencing methodologies to investigate how a model strain of E. faecalis discriminates its own DNA from foreign DNA, i.e., self versus nonself discrimination. We also assess the role of an E. faecalis genome modification system in modulating conjugative transfer of an antibiotic resistance plasmid. These results are significant because they demonstrate that differential genome modification impacts horizontal gene transfer frequencies in E. faecalis.

Dataset Information

Phased diploid genome assembly with single-molecule real-time sequencing.

Publications

Phased diploid genome assembly with single-molecule real-time sequencing.

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