The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Nicholas J Loman - One of the best experts on this subject based on the ideXlab platform.
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twenty years of Bacterial Genome sequencing
Nature Reviews Microbiology, 2015Co-Authors: Nicholas J Loman, Mark J PallenAbstract:Twenty years ago, the publication of the first Bacterial Genome sequence, from Haemophilus influenzae, shook the world of bacteriology. In this Timeline, we review the first two decades of Bacterial Genome sequencing, which have been marked by three revolutions: whole-Genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing. We summarize the social history of sequencing and its impact on our understanding of the biology, diversity and evolution of bacteria, while also highlighting spin-offs and translational impact in the clinic. We look forward to a 'sequencing singularity', where sequencing becomes the method of choice for as-yet unthinkable applications in bacteriology and beyond.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
Nature Methods, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:By error-correcting long nanopore reads and calling a consensus sequence using nanopore signal data, an entire Bacterial Genome is assembled de novo. We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
Nature Methods, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
bioRxiv, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:A method for de novo assembly of data from the Oxford Nanopore MinION instrument is presented which is able to reconstruct the sequence of an entire Bacterial chromosome in a single contig. Initially, overlaps between nanopore reads are detected. Reads are then subjected to one or more rounds of error correction by a multiple alignment process employing partial order graphs. After correction, reads are assembled using the Celera assembler. We show that this method is able to assemble nanopore reads from Escherichia coli K-12 MG1655 into a single contig of length 4.6Mb permitting a full reconstruction of gene order. The resulting assembly has 98.4% nucleotide identity compared to the finished reference Genome.
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a reference Bacterial Genome dataset generated on the minion portable single molecule nanopore sequencer
GigaScience, 2014Co-Authors: Joshua Quick, Aaron R Quinlan, Nicholas J LomanAbstract:Background The MinION™ is a new, portable single-molecule sequencer developed by Oxford Nanopore Technologies. It measures four inches in length and is powered from the USB 3.0 port of a laptop computer. The MinION™ measures the change in current resulting from DNA strands interacting with a charged protein nanopore. These measurements can then be used to deduce the underlying nucleotide sequence.
Jared T Simpson - One of the best experts on this subject based on the ideXlab platform.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
Nature Methods, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:By error-correcting long nanopore reads and calling a consensus sequence using nanopore signal data, an entire Bacterial Genome is assembled de novo. We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
Nature Methods, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
bioRxiv, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:A method for de novo assembly of data from the Oxford Nanopore MinION instrument is presented which is able to reconstruct the sequence of an entire Bacterial chromosome in a single contig. Initially, overlaps between nanopore reads are detected. Reads are then subjected to one or more rounds of error correction by a multiple alignment process employing partial order graphs. After correction, reads are assembled using the Celera assembler. We show that this method is able to assemble nanopore reads from Escherichia coli K-12 MG1655 into a single contig of length 4.6Mb permitting a full reconstruction of gene order. The resulting assembly has 98.4% nucleotide identity compared to the finished reference Genome.
Yonatan H Grad - One of the best experts on this subject based on the ideXlab platform.
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increased power from conditional Bacterial Genome wide association identifies macrolide resistance mutations in neisseria gonorrhoeae
Nature Communications, 2020Co-Authors: Kevin C, Tatum D Mortimer, Marissa A Duckett, Allison L Hicks, Nicole E Wheeler, Leonor Sanchezbuso, Yonatan H GradAbstract:The emergence of resistance to azithromycin complicates treatment of Neisseria gonorrhoeae, the etiologic agent of gonorrhea. Substantial azithromycin resistance remains unexplained after accounting for known resistance mutations. Bacterial Genome-wide association studies (GWAS) can identify novel resistance genes but must control for genetic confounders while maintaining power. Here, we show that compared to single-locus GWAS, conducting GWAS conditioned on known resistance mutations reduces the number of false positives and identifies a G70D mutation in the RplD 50S ribosomal protein L4 as significantly associated with increased azithromycin resistance (p-value = 1.08 × 10−11). We experimentally confirm our GWAS results and demonstrate that RplD G70D and other macrolide binding site mutations are prevalent (present in 5.42% of 4850 isolates) and widespread (identified in 21/65 countries across two decades). Overall, our findings demonstrate the utility of conditional associations for improving the performance of microbial GWAS and advance our understanding of the genetic basis of macrolide resistance. The mechanisms underlying resistance of Neisseria gonorrhoeae to the antibiotic azithromycin are incompletely understood. Here, Ma et al. conduct a conditional Genome-wide association study to identify new resistance mutations and experimentally confirm that a mutation in ribosomal protein L4 confers increased resistance.
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increased power from conditional Bacterial Genome wide association identifies macrolide resistance mutations in neisseria gonorrhoeae
Nature Communications, 2020Co-Authors: Kevin C, Tatum D Mortimer, Marissa A Duckett, Allison L Hicks, Nicole E Wheeler, Leonor Sanchezbuso, Yonatan H GradAbstract:The emergence of resistance to azithromycin complicates treatment of Neisseria gonorrhoeae, the etiologic agent of gonorrhea. Substantial azithromycin resistance remains unexplained after accounting for known resistance mutations. Bacterial Genome-wide association studies (GWAS) can identify novel resistance genes but must control for genetic confounders while maintaining power. Here, we show that compared to single-locus GWAS, conducting GWAS conditioned on known resistance mutations reduces the number of false positives and identifies a G70D mutation in the RplD 50S ribosomal protein L4 as significantly associated with increased azithromycin resistance (p-value = 1.08 × 10-11). We experimentally confirm our GWAS results and demonstrate that RplD G70D and other macrolide binding site mutations are prevalent (present in 5.42% of 4850 isolates) and widespread (identified in 21/65 countries across two decades). Overall, our findings demonstrate the utility of conditional associations for improving the performance of microbial GWAS and advance our understanding of the genetic basis of macrolide resistance.
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increased power from Bacterial Genome wide association conditional on known effects identifies neisseria gonorrhoeae macrolide resistance mutations in the 50s ribosomal protein l4
bioRxiv, 2020Co-Authors: Kevin C, Tatum D Mortimer, Marissa A Duckett, Allison L Hicks, Nicole E Wheeler, Leonor Sanchezbuso, Yonatan H GradAbstract:Abstract The emergence of resistance to azithromycin complicates treatment of N. gonorrhoeae, the etiologic agent of gonorrhea. Population genomic analyses of clinical isolates have demonstrated that some azithromycin resistance remains unexplained after accounting for the contributions of known resistance mutations in the 23S rRNA and the MtrCDE efflux pump. Bacterial Genome-wide association studies (GWAS) offer a promising approach for identifying novel resistance genes but must adequately address the challenge of controlling for genetic confounders while maintaining power to detect variants with lower effect sizes. Compared to a standard univariate GWAS, conducting GWAS conditioned on known resistance mutations with high effect sizes substantially reduced the number of variants that reached Genome-wide significance and identified a G70D mutation in the 50S ribosomal protein L4 (encoded by the gene rplD) as significantly associated with increased azithromycin minimum inhibitory concentrations (β = 1.03, 95% CI [0.76, 1.30]). The role and prevalence of these rplD mutations in conferring macrolide resistance in N. gonorrhoeae had been unclear. Here, we experimentally confirmed our GWAS results, identified other resistance-associated mutations in RplD, and showed that in total these RplD binding site mutations are prevalent (present in 5.42% of 4850 isolates) and geographically and temporally widespread (identified in 21/65 countries across two decades). Overall, our findings demonstrate the utility of conditional associations for improving the performance of microbial GWAS and advance our understanding of the genetic basis of macrolide resistance in a prevalent multidrug-resistant pathogen.
Joshua Quick - One of the best experts on this subject based on the ideXlab platform.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
Nature Methods, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:By error-correcting long nanopore reads and calling a consensus sequence using nanopore signal data, an entire Bacterial Genome is assembled de novo. We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
Nature Methods, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.
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a complete Bacterial Genome assembled de novo using only nanopore sequencing data
bioRxiv, 2015Co-Authors: Nicholas J Loman, Joshua Quick, Jared T SimpsonAbstract:A method for de novo assembly of data from the Oxford Nanopore MinION instrument is presented which is able to reconstruct the sequence of an entire Bacterial chromosome in a single contig. Initially, overlaps between nanopore reads are detected. Reads are then subjected to one or more rounds of error correction by a multiple alignment process employing partial order graphs. After correction, reads are assembled using the Celera assembler. We show that this method is able to assemble nanopore reads from Escherichia coli K-12 MG1655 into a single contig of length 4.6Mb permitting a full reconstruction of gene order. The resulting assembly has 98.4% nucleotide identity compared to the finished reference Genome.
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a reference Bacterial Genome dataset generated on the minion portable single molecule nanopore sequencer
GigaScience, 2014Co-Authors: Joshua Quick, Aaron R Quinlan, Nicholas J LomanAbstract:Background The MinION™ is a new, portable single-molecule sequencer developed by Oxford Nanopore Technologies. It measures four inches in length and is powered from the USB 3.0 port of a laptop computer. The MinION™ measures the change in current resulting from DNA strands interacting with a charged protein nanopore. These measurements can then be used to deduce the underlying nucleotide sequence.
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a reference Bacterial Genome dataset generated on the minion portable single molecule nanopore sequencer
GigaScience, 2014Co-Authors: Joshua Quick, Aaron R Quinlan, Nicholas J LomanAbstract:The MinION™ is a new, portable single-molecule sequencer developed by Oxford Nanopore Technologies. It measures four inches in length and is powered from the USB 3.0 port of a laptop computer. The MinION™ measures the change in current resulting from DNA strands interacting with a charged protein nanopore. These measurements can then be used to deduce the underlying nucleotide sequence. We present a read dataset from whole-Genome shotgun sequencing of the model organism Escherichia coli K-12 substr. MG1655 generated on a MinION™ device during the early-access MinION™ Access Program (MAP). Sequencing runs of the MinION™ are presented, one generated using R7 chemistry (released in July 2014) and one using R7.3 (released in September 2014). Base-called sequence data are provided to demonstrate the nature of data produced by the MinION™ platform and to encourage the development of customised methods for alignment, consensus and variant calling, de novo assembly and scaffolding. FAST5 files containing event data within the HDF5 container format are provided to assist with the development of improved base-calling methods.
Julian Parkhill - One of the best experts on this subject based on the ideXlab platform.
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comparison of Bacterial Genome assembly software for minion data and their applicability to medical microbiology
Microbial Genomics, 2016Co-Authors: Kim Judge, Martin Hunt, Sandra Reuter, Alan Tracey, Michael A Quail, Julian Parkhill, Sharon J PeacockAbstract:Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of Genome data, and relate this to the time taken to generate actionable information. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Evaluation of the four assemblies to represent the Genome structure revealed a single large inversion in the SPAdes assembly, which also incorrectly integrated a plasmid into the chromosomal contig. Almost 50 %, 80 % and 90 % of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. Using data from the first 6 h alone led to a less accurate, fragmented assembly, but data from the first 9 or 12 h generated similar assemblies to that from 48 h sequencing. Assemblies were generated in 2 h using Canu, indicating that going from isolate to assembled data is possible in less than 48 h. MinION data identified that genes responsible for resistance were carried by two plasmids encoding resistance to carbapenem and to sulphonamides, rifampicin and aminoglycosides, respectively.
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robust high throughput prokaryote de novo assembly and improvement pipeline for illumina data
Microbial genomics, 2016Co-Authors: Andrew J Page, Martin Hunt, Michael A Quail, Julian Parkhill, Nishadi De Silva, Simon R Harris, Thomas D Otto, Jacqueline A KeaneAbstract:The rapidly reducing cost of Bacterial Genome sequencing has lead to its routine use in large-scale microbial analysis. Though mapping approaches can be used to find differences relative to the reference, many bacteria are subject to constant evolutionary pressures resulting in events such as the loss and gain of mobile genetic elements, horizontal gene transfer through recombination and genomic rearrangements. De novo assembly is the reconstruction of the underlying Genome sequence, an essential step to understanding Bacterial Genome diversity. Here we present a high-throughput Bacterial assembly and improvement pipeline that has been used to generate nearly 20 000 annotated draft Genome assemblies in public databases. We demonstrate its performance on a public data set of 9404 Genomes. We find all the genes used in multi-locus sequence typing schema present in 99.6 % of assembled Genomes. When tested on low-, neutral- and high-GC organisms, more than 94 % of genes were present and completely intact. The pipeline has been proven to be scalable and robust with a wide variety of datasets without requiring human intervention. All of the software is available on GitHub under the GNU GPL open source license.
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comparison of Bacterial Genome assembly software for minion data
bioRxiv, 2016Co-Authors: Kim Judge, Martin Hunt, Sandra Reuter, Alan Tracey, Michael A Quail, Julian Parkhill, Sharon J PeacockAbstract:Antimicrobial resistance genes can be carried on plasmids or on mobile elements integrated into the chromosome. We sequenced a multidrug resistant Enterobacter kobei Genome isolated from wastewater in the United Kingdom, but were unable to conclusively identify plasmids from the short read assembly. Our aim was to compare and contrast the accuracy and characteristics of open source software (PBcR, Canu, miniasm and SPAdes) for the assembly of Bacterial Genomes (including plasmids) generated by the MinION instrument. Miniasm produced an assembly in the shortest time, but Canu produced the most accurate assembly overall. We found that MinION data alone was able to generate a contiguous and accurate assembly of an isolate with multiple plasmids.
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robust high throughput prokaryote de novo assembly and improvement pipeline for illumina data
bioRxiv, 2016Co-Authors: Andrew J Page, Martin Hunt, Michael A Quail, Julian Parkhill, Nishadi De Silva, Simon R Harris, Thomas D Otto, Jacqueline A KeaneAbstract:The rapidly reducing cost of Bacterial Genome sequencing has lead to its routine use in large scale microbial analysis. Though mapping approaches can be used to find differences relative to the reference, many bacteria are subject to constant evolutionary pressures resulting in events such as the loss and gain of mobile genetic elements, horizontal gene transfer through recombination and genomic rearrangements. De novo assembly is the reconstruction of the underlying Genome sequence, an essential step to understanding Bacterial Genome diversity. Here we present a high throughput Bacterial assembly and improvement pipeline that has been used to generate nearly 20,000 draft Genome assemblies in public databases. We demonstrate its performance on a public data set of 9,404 Genomes. We find all the genes used in MLST schema present in 99.6% of assembled Genomes. When tested on low, neutral and high GC organisms, more than 94% of genes were present and completely intact. The pipeline has proven to be scalable and robust with a wide variety of datasets without requiring human intervention. All of the software is available on GitHub under the GNU GPL open source license.
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microbiology in the post genomic era
Nature Reviews Microbiology, 2008Co-Authors: Duccio Medini, Julian Parkhill, Davide Serruto, David A Relman, Claudio Donati, Richard Moxon, Stanley Falkow, Rino RappuoliAbstract:Genomics has revolutionized every aspect of microbiology. Now, 13 years after the first Bacterial Genome was sequenced, it is important to pause and consider what has changed in microbiology research as a consequence of genomics. In this article, we review the evolving field of Bacterial typing and the genomic technologies that enable comparative analysis of multiple Genomes and the metaGenomes of complex microbial environments, and address the implications of the genomic era for the future of microbiology.