Methods and Software
The Microbial Genomics Methods and Software collection will bring together articles describing novel experimental, bioinformatics, modelling, and statistical approaches to the analysis of microbial genomics data, including databases or the integration of genomics with other data streams; as well as systematic comparisons or benchmarking of existing methodologies used in the field of microbial genomics. Guest-edited by Dr Zamin Iqbal (European Bioinformatics Institute) and Dr Caroline Colijn (Simon Fraser University), the collection aims to provide the microbial genomics community with new and systematically validated tools to advance their research.
The cover image for this collection brings together figures from two of retrospective articles in the collection: a phylogeny richly annotated with insertion sequence sites from the article on ISseeker by Adams et al. 2016 (bottom left); and a genome assembly graph from the article on completing bacterial genomes by Wick et al. 2017 (top right).
This collection is now open for submissions. Submit your article here, stating that your manuscript is part of the Methods and Software collection.
Collection Contents
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ClermonTyping: an easy-to-use and accurate in silico method for Escherichia genus strain phylotyping
More LessThe genus Escherichia is composed of Escherichia albertii, E. fergusonii, five cryptic Escherichia clades and E. coli sensu stricto. Furthermore, the E. coli species can be divided into seven main phylogroups termed A, B1, B2, C, D, E and F. As specific lifestyles and/or hosts can be attributed to these species/phylogroups, their identification is meaningful for epidemiological studies. Classical phenotypic tests fail to identify non-sensu stricto E. coli as well as phylogroups. Clermont and colleagues have developed PCR assays that allow the identification of most of these species/phylogroups, the triplex/quadruplex PCR for E. coli phylogroup determination being the most popular. With the growing availability of whole genome sequences, we have developed the ClermonTyping method and its associated web-interface, the ClermonTyper, that allows a given strain sequence to be assigned to E. albertii, E. fergusonii, Escherichia clades I–V, E. coli sensu stricto as well as to the seven main E. coli phylogroups. The ClermonTyping is based on the concept of in vitro PCR assays and maintains the principles of ease of use and speed that prevailed during the development of the in vitro assays. This in silico approach shows 99.4 % concordance with the in vitro PCR assays and 98.8 % with the Mash genome-clustering tool. The very few discrepancies result from various errors occurring mainly from horizontal gene transfers or SNPs in the primers. We propose the ClermonTyper as a freely available resource to the scientific community at: http://clermontyping.iame-research.center/.
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chewBBACA: A complete suite for gene-by-gene schema creation and strain identification
Gene-by-gene approaches are becoming increasingly popular in bacterial genomic epidemiology and outbreak detection. However, there is a lack of open-source scalable software for schema definition and allele calling for these methodologies. The chewBBACA suite was designed to assist users in the creation and evaluation of novel whole-genome or core-genome gene-by-gene typing schemas and subsequent allele calling in bacterial strains of interest. chewBBACA performs the schema creation and allele calls on complete or draft genomes resulting from de novo assemblers. The chewBBACA software uses Python 3.4 or higher and can run on a laptop or in high performance clusters making it useful for both small laboratories and large reference centers. ChewBBACA is available at https://github.com/B-UMMI/chewBBACA.
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Comprehensive assessment of the quality of Salmonella whole genome sequence data available in public sequence databases using the Salmonella in silico Typing Resource (SISTR)
Public health and food safety institutions around the world are adopting whole genome sequencing (WGS) to replace conventional methods for characterizing Salmonella for use in surveillance and outbreak response. Falling costs and increased throughput of WGS have resulted in an explosion of data, but questions remain as to the reliability and robustness of the data. Due to the critical importance of serovar information to public health, it is essential to have reliable serovar assignments available for all of the Salmonella records. The current study used a systematic assessment and curation of all Salmonella in the sequence read archive (SRA) to assess the state of the data and their utility. A total of 67 758 genomes were assembled de novo and quality-assessed for their assembly metrics as well as species and serovar assignments. A total of 42 400 genomes passed all of the quality criteria but 30.16 % of genomes were deposited without serotype information. These data were used to compare the concordance of reported and predicted serovars for two in silico prediction tools, multi-locus sequence typing (MLST) and the Salmonella in silico Typing Resource (SISTR), which produced predictions that were fully concordant with 87.51 and 91.91 % of the tested isolates, respectively. Concordance of in silico predictions increased when serovar variants were grouped together, 89.25 % for MLST and 94.98 % for SISTR. This study represents the first large-scale validation of serovar information in public genomes and provides a large validated set of genomes, which can be used to benchmark new bioinformatics tools.
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Coupling next-generation sequencing to dominant positive screens for finding antibiotic cellular targets and resistance mechanisms in Escherichia coli
More LessIn order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.
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Culture-independent approaches to chlamydial genomics
More LessThe expanding field of bacterial genomics has revolutionized our understanding of microbial diversity, biology and phylogeny. For most species, DNA extracted from culture material is used as the template for genome sequencing; however, the majority of microbes are actually uncultivable, and others, such as obligate intracellular bacteria, require laborious tissue culture to yield sufficient genomic material for sequencing. Chlamydiae are one such group of obligate intracellular microbes whose characterization has been hampered by this requirement. To circumvent these challenges, researchers have developed culture-independent sample preparation methods that can be applied to the sample directly or to genomic material extracted from the sample. These methods, which encompass both targeted [immunomagnetic separation-multiple displacement amplification (IMS-MDA) and sequence capture] and non-targeted approaches (host methylated DNA depletion-microbial DNA enrichment and cell-sorting-MDA), have been applied to a range of clinical and environmental samples to generate whole genomes of novel chlamydial species and strains. This review aims to provide an overview of the application, advantages and limitations of these targeted and non-targeted approaches in the chlamydial context. The methods discussed also have broad application to other obligate intracellular bacteria or clinical and environmental samples.
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Completing bacterial genome assemblies with multiplex MinION sequencing
More LessIllumina sequencing platforms have enabled widespread bacterial whole genome sequencing. While Illumina data is appropriate for many analyses, its short read length limits its ability to resolve genomic structure. This has major implications for tracking the spread of mobile genetic elements, including those which carry antimicrobial resistance determinants. Fully resolving a bacterial genome requires long-read sequencing such as those generated by Oxford Nanopore Technologies (ONT) platforms. Here we describe our use of the ONT MinION to sequence 12 isolates of Klebsiella pneumoniae on a single flow cell. We assembled each genome using a combination of ONT reads and previously available Illumina reads, and little to no manual intervention was needed to achieve fully resolved assemblies using the Unicycler hybrid assembler. Assembling only ONT reads with Canu was less effective, resulting in fewer resolved genomes and higher error rates even following error correction with Nanopolish. We demonstrate that multiplexed ONT sequencing is a valuable tool for high-throughput bacterial genome finishing. Specifically, we advocate the use of Illumina sequencing as a first analysis step, followed by ONT reads as needed to resolve genomic structure.
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Comparative scaffolding and gap filling of ancient bacterial genomes applied to two ancient Yersinia pestis genomes
More Lessdoi: 10.1099/mgen.0.000123.001.
Yersinia pestis is the causative agent of the bubonic plague, a disease responsible for several dramatic historical pandemics. Progress in ancient DNA (aDNA) sequencing rendered possible the sequencing of whole genomes of important human pathogens, including the ancient Y. pestis strains responsible for outbreaks of the bubonic plague in London in the 14th century and in Marseille in the 18th century, among others. However, aDNA sequencing data are still characterized by short reads and non-uniform coverage, so assembling ancient pathogen genomes remains challenging and often prevents a detailed study of genome rearrangements. It has recently been shown that comparative scaffolding approaches can improve the assembly of ancient Y. pestis genomes at a chromosome level. In the present work, we address the last step of genome assembly, the gap-filling stage. We describe an optimization-based method AGapEs (ancestral gap estimation) to fill in inter-contig gaps using a combination of a template obtained from related extant genomes and aDNA reads. We show how this approach can be used to refine comparative scaffolding by selecting contig adjacencies supported by a mix of unassembled aDNA reads and comparative signal. We applied our method to two Y. pestis data sets from the London and Marseilles outbreaks, for which we obtained highly improved genome assemblies for both genomes, comprised of, respectively, five and six scaffolds with 95 % of the assemblies supported by ancient reads. We analysed the genome evolution between both ancient genomes in terms of genome rearrangements, and observed a high level of synteny conservation between these strains.
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Comparison of classical multi-locus sequence typing software for next-generation sequencing data
Multi-locus sequence typing (MLST) is a widely used method for categorizing bacteria. Increasingly, MLST is being performed using next-generation sequencing (NGS) data by reference laboratories and for clinical diagnostics. Many software applications have been developed to calculate sequence types from NGS data; however, there has been no comprehensive review to date on these methods. We have compared eight of these applications against real and simulated data, and present results on: (1) the accuracy of each method against traditional typing methods, (2) the performance on real outbreak datasets, (3) the impact of contamination and varying depth of coverage, and (4) the computational resource requirements.
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CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online resource for the medical microbiology community
The increasing availability and decreasing cost of high-throughput sequencing has transformed academic medical microbiology, delivering an explosion in available genomes while also driving advances in bioinformatics. However, many microbiologists are unable to exploit the resulting large genomics datasets because they do not have access to relevant computational resources and to an appropriate bioinformatics infrastructure. Here, we present the Cloud Infrastructure for Microbial Bioinformatics (CLIMB) facility, a shared computing infrastructure that has been designed from the ground up to provide an environment where microbiologists can share and reuse methods and data.
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Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology
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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