Estimating coverage in metagenomic data sets and why it matters

Rodriguez-R, Luis M.; Konstantinidis, Konstantinos T.

doi:10.1038/ismej.2014.76

Cited by 151 publications

(121 citation statements)

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“…S1 shows our complete bioinformatics pipeline for all analyses performed). We used estimates of shotgun sequencing coverage to evaluate our metagenomic data sets for potential pathogen recovery using Nonpareil, an assembly-and reference-independent measure of overlap among sequencing reads (11). Although the total microbial sequencing recovery varied between samples, Ͼ95% of the microbial population was covered in 7 (63%) of the metagenomes, according to our estimates (Fig.…”

Section: Figmentioning

confidence: 99%

Metagenomics of Two Severe Foodborne Outbreaks Provides Diagnostic Signatures and Signs of Coinfection Not Attainable by Traditional Methods

Huang

Luo

Pena‐Gonzalez

et al. 2017

Appl Environ Microbiol

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Diagnostic testing for foodborne pathogens relies on culture-based techniques that are not rapid enough for real-time disease surveillance and do not give a quantitative picture of pathogen abundance or the response of the natural microbiome. Powerful sequence-based culture-independent approaches, such as shotgun metagenomics, could sidestep these limitations and potentially reveal a pathogenspecific signature on the microbiome that would have implications not only for diagnostics but also for better understanding disease progression and pathogen ecology. However, metagenomics have not yet been validated for foodborne pathogen detection. Toward closing these gaps, we applied shotgun metagenomics to stool samples collected from two geographically isolated (Alabama and Colorado) foodborne outbreaks, where the etiologic agents were identified by culture-dependent methods as distinct strains of Salmonella enterica subsp. enterica serovar Heidelberg. Metagenomic investigations were consistent with the culture-based findings and revealed, in addition, the in situ abundance and level of intrapopulation diversity of the pathogen, the possibility of coinfections with Staphylococcus aureus, overgrowth of commensal Escherichia coli, and significant shifts in the gut microbiome during infection relative to reference healthy samples. Additionally, we designed our bioinformatics pipeline to deal with several challenges associated with the analysis of clinical samples, such as the high frequency of coeluting human DNA sequences and assessment of the virulence potential of pathogens. Comparisons of these results to those of other studies revealed that in several, but not all, cases of diarrheal outbreaks, the disease and healthy states of the gut microbial community might be distinguishable, opening new possibilities for diagnostics.IMPORTANCE Diagnostic testing for enteric pathogens has relied for decades on culture-based techniques, but a total of 38.4 million cases of foodborne illness per year cannot be attributed to specific causes. This study describes new cultureindependent metagenomic approaches and the associated bioinformatics pipeline to detect and type the causative agents of microbial disease with unprecedented accuracy, opening new possibilities for the future development of health technologies and diagnostics. Our tools and approaches should be applicable to other microbial diseases in addition to foodborne diarrhea.KEYWORDS Salmonella, diagnostics, diarrhea, human gut, metagenomics D iagnostic testing for enteric pathogens has relied for decades on culture-based techniques, and culture-derived isolates currently form the foundation for public health surveillance. However, the clinical landscape is rapidly changing as culture-

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Section: Figmentioning

confidence: 99%

Metagenomics of Two Severe Foodborne Outbreaks Provides Diagnostic Signatures and Signs of Coinfection Not Attainable by Traditional Methods

Huang

Luo

Pena‐Gonzalez

et al. 2017

Appl Environ Microbiol

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“…Rodriguez-R and Konstantinidis, 2014a). The July and October 2010 samples (oiled and weathered oil) were assembled into B56 000 contigs per sample with N50 of B1400 bp; while those from recovered samples resulted in B12 000 contigs per sample with N50 of B745 bp (Supplementary File S1).…”

Section: Description Of Samples and Their Metagenomesmentioning

confidence: 99%

Microbial community successional patterns in beach sands impacted by the Deepwater Horizon oil spill

et al. 2015

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Although petroleum hydrocarbons discharged from the Deepwater Horizon (DWH) blowout were shown to have a pronounced impact on indigenous microbial communities in the Gulf of Mexico, effects on nearshore or coastal ecosystems remain understudied. This study investigated the successional patterns of functional and taxonomic diversity for over 1 year after the DWH oil was deposited on Pensacola Beach sands (FL, USA), using metagenomic and 16S rRNA gene amplicon techniques. Gamma- and Alphaproteobacteria were enriched in oiled sediments, in corroboration of previous studies. In contrast to previous studies, we observed an increase in the functional diversity of the community in response to oil contamination and a functional transition from generalist populations within 4 months after oil came ashore to specialists a year later, when oil was undetectable. At the latter time point, a typical beach community had reestablished that showed little to no evidence of oil hydrocarbon degradation potential, was enriched in archaeal taxa known to be sensitive to xenobiotics, but differed significantly from the community before the oil spill. Further, a clear succession pattern was observed, where early responders to oil contamination, likely degrading aliphatic hydrocarbons, were replaced after 3 months by populations capable of aromatic hydrocarbon decomposition. Collectively, our results advance the understanding of how natural benthic microbial communities respond to crude oil perturbation, supporting the specialization-disturbance hypothesis; that is, the expectation that disturbance favors generalists, while providing (microbial) indicator species and genes for the chemical evolution of oil hydrocarbons during degradation and weathering.

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“…S5), meaning that at least 55% (for 2,4-D mesocosms, coverage was Ͼ73%) of the total extracted unique (nonredundant) DNA was sequenced. This level represents an appropriate coverage for statistically robust comparisons of gene and species relative abundance between metagenomic data sets (35). Based on both MyTaxa taxonomic assignments of metagenomic contigs and QIIME analysis of 16S rRNA gene fragments contained in the metagenomic reads, the community diversity of the 2,4-D mesocosms was much higher at the beginning than at later time points, presumably due to the strong selection pressure of the substrate and death due to restricted growth conditions (e.g., lack of primary production, 2,4-D toxicity to some community members).…”

Section: Figmentioning

confidence: 99%

Quantifying the Importance of the Rare Biosphere for Microbial Community Response to Organic Pollutants in a Freshwater Ecosystem

Wang

Hatt

Tsementzi

et al. 2017

Appl Environ Microbiol

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A single liter of water contains hundreds, if not thousands, of bacterial and archaeal species, each of which typically makes up a very small fraction of the total microbial community (Ͻ0.1%), the so-called "rare biosphere." How often, and via what mechanisms, e.g., clonal amplification versus horizontal gene transfer, the rare taxa and genes contribute to microbial community response to environmental perturbations represent important unanswered questions toward better understanding the value and modeling of microbial diversity. We tested whether rare species frequently responded to changing environmental conditions by establishing 20-liter planktonic mesocosms with water from Lake Lanier (Georgia, USA) and perturbing them with organic compounds that are rarely detected in the lake, including 2,4-dichlorophenoxyacetic acid (2,4-D), 4-nitrophenol (4-NP), and caffeine. The populations of the degraders of these compounds were initially below the detection limit of quantitative PCR (qPCR) or metagenomic sequencing methods, but they increased substantially in abundance after perturbation. Sequencing of several degraders (isolates) and time-series metagenomic data sets revealed distinct cooccurring alleles of degradation genes, frequently carried on transmissible plasmids, especially for the 2,4-D mesocosms, and distinct species dominating the post-enrichment microbial communities from each replicated mesocosm. This diversity of species and genes also underlies distinct degradation profiles among replicated mesocosms. Collectively, these results supported the hypothesis that the rare biosphere can serve as a genetic reservoir, which can be frequently missed by metagenomics but enables community response to changing environmental conditions caused by organic pollutants, and they provided insights into the size of the pool of rare genes and species.IMPORTANCE A single liter of water or gram of soil contains hundreds of lowabundance bacterial and archaeal species, the so called rare biosphere. The value of this astonishing biodiversity for ecosystem functioning remains poorly understood, primarily due to the fact that microbial community analysis frequently focuses on abundant organisms. Using a combination of culture-dependent and culture-independent (metagenomics) techniques, we showed that rare taxa and genes commonly contribute to the microbial community response to organic pollutants. Our findings should have implications for future studies that aim to study the role of rare species in environmental processes, including environmental bioremediation efforts of oil spills or other contaminants.

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Estimating coverage in metagenomic data sets and why it matters

Cited by 151 publications

References 10 publications

Metagenomics of Two Severe Foodborne Outbreaks Provides Diagnostic Signatures and Signs of Coinfection Not Attainable by Traditional Methods

Metagenomics of Two Severe Foodborne Outbreaks Provides Diagnostic Signatures and Signs of Coinfection Not Attainable by Traditional Methods

Microbial community successional patterns in beach sands impacted by the Deepwater Horizon oil spill

Quantifying the Importance of the Rare Biosphere for Microbial Community Response to Organic Pollutants in a Freshwater Ecosystem

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