Methods and Software
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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
41 - 49 of 49 results
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Robust high-throughput prokaryote de novo assembly and improvement pipeline for Illumina data
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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Quantitative assessment of insertion sequence impact on bacterial genome architecture
More LessInsertion sequence (IS) elements are important mediators of genome plasticity and can lead to phenotypic changes with evolutionary significance. In multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae, IS elements have contributed significantly to the mobilization of genes that encode resistance to antimicrobial drugs. A systematic analysis of IS elements is needed for a more comprehensive understanding of their evolutionary impact. We developed a computational approach (ISseeker) to annotate IS elements in draft genome assemblies and applied the method to analysis of IS elements in all publicly available A. baumannii(>1000) and K. pneumoniae(>800) genome sequences, in a phylogenetic context. Most IS elements in A. baumanniigenomes are species-specific ISAba elements, whereas K. pneumoniaegenomes contain significant numbers of both ISKpn elements and elements that are found throughout the Enterobacteriaceae. A. baumanniigenomes have a higher density of IS elements than K. pneumoniae, averaging ~33 vs ~27 copies per genome. In K. pneumoniae, several insertion sites are shared by most genomes in the ST258 clade, whereas in A. baumannii, different IS elements are abundant in different phylogenetic groups, even among closely related Global Clone 2 strains. IS elements differ in the distribution of insertion locations relative to genes, with some more likely to disrupt genes and others predominantly in intergenic regions. Several genes and intergenic regions had multiple independent insertion events, suggesting that those events may confer a selective advantage. Genome- and taxon-wide characterization of insertion locations revealed that IS elements have been active contributors to genome diversity in both species.
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Declaring a tuberculosis outbreak over with genomic epidemiology
We report an updated method for inferring the time at which an infectious disease was transmitted between persons from a time-labelled pathogen genome phylogeny. We applied the method to 48 Mycobacterium tuberculosis genomes as part of a real-time public health outbreak investigation, demonstrating that although active tuberculosis (TB) cases were diagnosed through 2013, no transmission events took place beyond mid-2012. Subsequent cases were the result of progression from latent TB infection to active disease, and not recent transmission. This evolutionary genomic approach was used to declare the outbreak over in January 2015.
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SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments
Rapidly decreasing genome sequencing costs have led to a proportionate increase in the number of samples used in prokaryotic population studies. Extracting single nucleotide polymorphisms (SNPs) from a large whole genome alignment is now a routine task, but existing tools have failed to scale efficiently with the increased size of studies. These tools are slow, memory inefficient and are installed through non-standard procedures. We present SNP-sites which can rapidly extract SNPs from a multi-FASTA alignment using modest resources and can output results in multiple formats for downstream analysis. SNPs can be extracted from a 8.3 GB alignment file (1842 taxa, 22 618 sites) in 267 seconds using 59 MB of RAM and 1 CPU core, making it feasible to run on modest computers. It is easy to install through the Debian and Homebrew package managers, and has been successfully tested on more than 20 operating systems. SNP-sites is implemented in C and is available under the open source license GNU GPL version 3.
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PhagePhisher: a pipeline for the discovery of covert viral sequences in complex genomic datasets
More LessObtaining meaningful viral information from large sequencing datasets presents unique challenges distinct from prokaryotic and eukaryotic sequencing efforts. The difficulties surrounding this issue can be ascribed in part to the genomic plasticity of viruses themselves as well as the scarcity of existing information in genomic databases. The open-source software PhagePhisher (http://www.putonti-lab.com/phagephisher) has been designed as a simple pipeline to extract relevant information from complex and mixed datasets, and will improve the examination of bacteriophages, viruses, and virally related sequences, in a range of environments. Key aspects of the software include speed and ease of use; PhagePhisher can be used with limited operator knowledge of bioinformatics on a standard workstation. As a proof-of-concept, PhagePhisher was successfully implemented with bacteria–virus mixed samples of varying complexity. Furthermore, viral signals within microbial metagenomic datasets were easily and quickly identified by PhagePhisher, including those from prophages as well as lysogenic phages, an important and often neglected aspect of examining phage populations in the environment. PhagePhisher resolves viral-related sequences which may be obscured by or imbedded in bacterial genomes.
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SimBac: simulation of whole bacterial genomes with homologous recombination
More LessBacteria can exchange genetic material, or acquire genes found in the environment. This process, generally known as bacterial recombination, can have a strong impact on the evolution and phenotype of bacteria, for example causing the spread of antibiotic resistance across clades and species, but can also disrupt phylogenetic and transmission inferences. With the increasing affordability of whole genome sequencing, the need has emerged for an efficient simulator of bacterial evolution to test and compare methods for phylogenetic and population genetic inference, and for simulation-based estimation. We present SimBac, a whole-genome bacterial evolution simulator that is roughly two orders of magnitude faster than previous software and includes a more general model of bacterial evolution, allowing both within- and between-species homologous recombination. Since methods modelling bacterial recombination generally focus on only one of these two modes of recombination, the possibility to simulate both allows for a general and fair benchmarking. SimBac is available from https://github.com/tbrown91/SimBac and is distributed as open source under the terms of the GNU General Public Licence.
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Recombination produces coherent bacterial species clusters in both core and accessory genomes
More LessBackground:Population samples show bacterial genomes can be divided into a core of ubiquitous genes and accessory genes that are present in a fraction of isolates. The ecological significance of this variation in gene content remains unclear. However, microbiologists agree that a bacterial species should be ‘genomically coherent’, even though there is no consensus on how this should be determined.
Results:We use a parsimonious model combining diversification in both the core and accessory genome, including mutation, homologous recombination (HR) and horizontal gene transfer (HGT) introducing new loci, to produce a population of interacting clusters of strains with varying genome content. New loci introduced by HGT may then be transferred on by HR. The model fits well to a systematic population sample of 616 pneumococcal genomes, capturing the major features of the population structure with parameter values that agree well with empirical estimates.
Conclusions:The model does not include explicit selection on individual genes, suggesting that crude comparisons of gene content may be a poor predictor of ecological function. We identify a clearly divergent subpopulation of pneumococci that are inconsistent with the model and may be considered genomically incoherent with the rest of the population. These strains have a distinct disease tropism and may be rationally defined as a separate species. We also find deviations from the model that may be explained by recent population bottlenecks or spatial structure.
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Shetti, a simple tool to parse, manipulate and search large datasets of sequences
More LessParsing and manipulating long and/or multiple protein or gene sequences can be a challenging process for experimental biologists and microbiologists lacking prior knowledge of bioinformatics and programming. Here we present a simple, easy, user-friendly and versatile tool to parse, manipulate and search within large datasets of long and multiple protein or gene sequences. The Shetti tool can be used to search for a sequence, species, protein/gene or pattern/motif. Moreover, it can also be used to construct a universal consensus or molecular signatures for proteins based on their physical characteristics. Shetti is an efficient and fast tool that can deal with large sets of long sequences efficiently. Shetti parses UniProt Knowledgebase and NCBI GenBank flat files and visualizes them as a table.
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EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens
More LessRecent development of CRISPR-Cas9 genome editing has enabled highly efficient and versatile manipulation of a variety of organisms and adaptation of the CRISPR-Cas9 system to eukaryotic pathogens has opened new avenues for studying these otherwise hard to manipulate organisms. Here we describe a webtool, Eukaryotic Pathogen gRNA Design Tool (EuPaGDT; available at http://grna.ctegd.uga.edu), which identifies guide RNA (gRNA) in input gene(s) to guide users in arriving at well-informed and appropriate gRNA design for many eukaryotic pathogens. Flexibility in gRNA design, accommodating unique eukaryotic pathogen (gene and genome) attributes and high-throughput gRNA design are the main features that distinguish EuPaGDT from other gRNA design tools. In addition to employing an array of known principles to score and rank gRNAs, EuPaGDT implements an effective on-target search algorithm to identify gRNA targeting multi-gene families, which are highly represented in these pathogens and play important roles in host–pathogen interactions. EuPaGDT also identifies and scores microhomology sequences flanking each gRNA targeted cut-site; these sites are often essential for the microhomology-mediated end joining process used for double-stranded break repair in these organisms. EuPaGDT also assists users in designing single-stranded oligonucleotides for homology directed repair. In batch processing mode, EuPaGDT is able to process genome-scale sequences, enabling preparation of gRNA libraries for large-scale screening projects.
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