Genomic diversity affects the accuracy of bacterial SNP calling pipelines

Bush, Stephen J.; Foster, Dona; Eyre, David W; Clark, Emily L.; Maio, Nicola De; Shaw, Liam P.; Stoesser, Nicole; Peto, Tim; Crook, Derrick W.; Walker, A Sarah

doi:10.1101/653774

Cited by 11 publications

(11 citation statements)

References 90 publications

(121 reference statements)

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“…genus-level phylogeny) or genera within a single tree, nor have any been tested on microbial eukaryotic genomes. In addition, most of these tools require users to select a reference genome, which can have dramatic impact on the alignments and resulting SNP calls 26 , and are unable to distinguish or map SNPs to their functional annotation, and hence cannot perform molecular evolution analysis.…”

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confidence: 99%

Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life

Shakya

Ahmed

Davenport

et al. 2020

Sci Rep

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chien-chi Lo & patrick S. G. chain * there is growing interest in reconstructing phylogenies from the copious amounts of genome sequencing projects that target related viral, bacterial or eukaryotic organisms. to facilitate the construction of standardized and robust phylogenies for disparate types of projects, we have developed a complete bioinformatic workflow, with a web-based component to perform phylogenetic and molecular evolutionary (phaMe) analysis from sequencing reads, draft assemblies or completed genomes of closely related organisms. furthermore, the ability to incorporate raw data, including some metagenomic samples containing a target organism (e.g. from clinical samples with suspected infectious agents), shows promise for the rapid phylogenetic characterization of organisms within complex samples without the need for prior assembly. The reconstruction of organismal evolutionary history using phylogenetics is a fundamental method applied to many areas of biology. Single nucleotide polymorphisms (SNPs), one of the dominant forms of evolutionary change, have become an indispensable tool for phylogenetic analyses 1-4. Phylogenies in the pre-genomic era relied on SNPs and conserved sites within a single locus, and was later extended to multiple loci, such as in multiple locus sequence typing (MLST). Although still valuable, these methods only consider evolutionary signals originating within a small fraction of the genome, are unable to capture the complete variation within species, and generally provide a weak phylogenetic signal, particularly within a species, and do not always reflect the true evolutionary history of species 5. While phylogenetic analyses that use many conserved genes (orthologs) are a great improvement, these methods require annotated coding regions, whose predictions are not always accurate or available 6. Furthermore, they are impacted by horizontal gene transfer (HGT) 7 , recombination 8 , rate heterogeneity 9 , and incomplete lineage sorting. Genome-wide SNPs are one of the best measures of phylogenetic diversity as they can discriminate among closely related organisms and help resolve both short and long branches in a tree 10,11. Since selectively neutral SNPs accumulate at a uniform rate, they can be used to measure divergence between species as well as strains 12,13. Furthermore, due to the large number of SNPs found along the length of entire genomes, the use of whole-genome SNPs minimizes the impact of random sequencing and assembly errors that can impact individual loci, as well as biases due to individual genes under strong selective pressure. Some inherent biases remain with whole genome SNP approaches that are similar to loci-based phylogenies such as HGT, recombination, and rate heterogeneity. Although genome-wide sequencing now allows examination of the full complement of genomic variation, the number of completed and finished genomes are increasingly falling behind the generation of new draft genomes, due to the lack of computational or other resources. For example, o...

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confidence: 99%

Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life

Shakya

Ahmed

Davenport

et al. 2020

Sci Rep

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“…coli and Klebsiella spp. [37], together with NCBI canonical reference sequences for several pathogen species expected to be absent from sink drain microbiota (; left). This demonstrated similar sink, ward and temporal structure to that of culture, particularly underlying similarities in the microbiota of nearby sinks, as well as flux between sampling timepoints.…”

Section: Resultsmentioning

confidence: 99%

Genomic surveillance of Escherichia coli and Klebsiella spp. in hospital sink drains and patients

et al. 2020

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Escherichia coli and Klebsiella spp. are important human pathogens that cause a wide spectrum of clinical disease. In healthcare settings, sinks and other wastewater sites have been shown to be reservoirs of antimicrobial-resistant E. coli and Klebsiella spp., particularly in the context of outbreaks of resistant strains amongst patients. Without focusing exclusively on resistance markers or a clinical outbreak, we demonstrate that many hospital sink drains are abundantly and persistently colonized with diverse populations of E. coli, Klebsiella pneumoniae and Klebsiella oxytoca, including both antimicrobial-resistant and susceptible strains. Using whole-genome sequencing of 439 isolates, we show that environmental bacterial populations are largely structured by ward and sink, with only a handful of lineages, such as E. coli ST635, being widely distributed, suggesting different prevailing ecologies, which may vary as a result of different inputs and selection pressures. Whole-genome sequencing of 46 contemporaneous patient isolates identified one (2 %; 95 % CI 0.05-11 %) E. coli urine infection-associated isolate with high similarity to a prior sink isolate, suggesting that sinks may contribute to up to 10 % of infections caused by these organisms in patients on the ward over the same timeframe. Using metagenomics from 20 sink-timepoints, we show that sinks also harbour many clinically relevant antimicrobial resistance genes including bla CTX-M , bla SHV and mcr, and may act as niches for the exchange and amplification of these genes. Our study reinforces the potential role of sinks in contributing to Enterobacterales infection and antimicrobial resistance in hospital patients, something that could be amenable to intervention. This article contains data hosted by Microreact. DATA SummARyRaw sequencing data are available under NCBI SRA accessions PRJNA604910 and PRJNA604975 (cultured isolates), and ENA project PRJEB36775 (metagenomes). A supporting data repository containing metadata, phylogenies, Jupyter notebooks and Pavian reports is archived at Figshare (doi:10.6084/m9.figshare.12337283). Microreact projects are available for E. coli, K. oxytoca and K. pneumoniae cultures. The authors confirm all supporting data, code and protocols have been provided within the article or the supporting data repository.

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“…There is a need for these standards to be implemented to help harmonize the analysis and the consistency in the interpretation of WGS sequence data [29], [30]. It is also critical due to the various potential sources for error in sequencing and variant-calling processes [31], [32]. To optimise the quality of the data used to generate variant calls, many of the recommendations and best practises used are shared between human and microbial genomic NGS assays [33], [34].…”

Section: Comparison Of the Fastq Data Set Submitted By Laboratories mentioning

confidence: 99%

Proficiency Testing for bacterial whole genome sequencing in assuring the quality of microbiology diagnostics in clinical and public health laboratories

Lau¹,

Silva

Theis³

et al. 2020

Preprint

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The adoption of whole genome sequencing (WGS) data over the past decade for pathogen surveillance, and decision-making for infectious diseases has rapidly transformed the landscape of clinical microbiology and public health. However, for successful transition to routine use of these techniques, it is crucial to ensure the WGS data generated meet defined quality standards for pathogen identification, typing, antimicrobial resistance detection and surveillance. Further, the ongoing development of these standards will ensure that the bioinformatic processes are capable of accurately identifying and characterising organisms of interest, and thereby facilitate the integration of WGS into routine clinical and public health laboratory setting. A pilot proficiency testing (PT) program for WGS of infectious agents was developed to facilitate widely applicable standardisation and benchmarking standards for WGS across a range of laboratories. The PT participating laboratories were required to generate WGS data from two bacterial isolates, and submit the raw data for independent bioinformatics analysis, as well as analyse the data with their own processes and answer relevant questions about the data. Overall, laboratories used a diverse range of bioinformatics tools and could generate and analyse high-quality data, either meeting or exceeding the minimum requirements. This pilot has provided valuable insight into the current state of genomics in clinical microbiology and public health laboratories across Australia. It will provide a baseline guide for the standardisation of WGS and enable the development of a PT program that allows an ongoing performance benchmark for accreditation of WGS-based test processes.

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Genomic diversity affects the accuracy of bacterial SNP calling pipelines

Cited by 11 publications

References 90 publications

Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life

Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life

Genomic surveillance of Escherichia coli and Klebsiella spp. in hospital sink drains and patients

Proficiency Testing for bacterial whole genome sequencing in assuring the quality of microbiology diagnostics in clinical and public health laboratories

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