Crass: identification and reconstruction of CRISPR from unassembled metagenomic data

Skennerton, Connor T.; Imelfort, Michael; Tyson, Gene W.

doi:10.1093/nar/gkt183

Cited by 152 publications

(135 citation statements)

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“…Crass was used to identify CRISPR repeat and spacer sequences 49 , and was run both on individual sample reads and combined reads from all samples. To identify the microbial hosts of viruses and determine if viral predation was ongoing in the deep shale, we used BLASTn with an E-value cutoff of 1e-8 to identify contigs with repeat and spacers.…”

Section: Methodsmentioning

confidence: 99%

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

et al. 2016

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Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here, the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that many of the persisting organisms play roles in methylamine cycling, ultimately supporting methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while thiosulfate reduction could produce sulfide, contributing to reservoir souring and infrastructure corrosion. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface. S hale gas accounts for one-third of natural gas energy resources worldwide. It has been estimated that shale gas will provide half of the natural gas in the USA, annually, by 2040, with the Marcellus shale in the Appalachian Basin projected to produce three times more than any other formation 1 . Recovery of these hydrocarbons is dependent on hydraulic fracturing technologies, where the high-pressure injection of water and chemical additives generates extensive fractures in the shale matrix. Hydrocarbons trapped in tiny pore spaces are subsequently released and collected at the wellpad surface, together with a portion of the injected fluids that have reacted with the shale formation. The mixture of injected fluids and hydrocarbons collected is referred to as 'produced fluids'.Microbial metabolism and growth in hydrocarbon reservoirs has both positive and negative impacts on energy recovery. Whereas stimulation of methanogens in coal beds enhances energy recovery 2 , bacterial hydrogen sulfide production ('reservoir souring') decreases profits and contributes to corrosion and the risk of environmental contamination 3 . Additionally, biomass accumulation within newly generated fractures may reduce their permeability, decreasing natural gas recovery. Despite these potential microbial impacts, little is known about the function and activity of microorganisms in hydraulically fractured shale.Initial work by our group and others 4-9 used single marker gene analyses to identify microorganisms from several geographically distinct shale formations. These analyses showed similar halotolerant taxa in produced fluids several months after hydraulic fracturing. To assign functional roles to these organisms, we conducted metagenomic and metabolite analyses on input and produced fluids up to a year after hydraulic fracturing (HF) from two Appala...

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Section: Methodsmentioning

confidence: 99%

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

et al. 2016

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“…Because CRISPR arrays’ repetitive nature makes them highly difficult to assemble, CRISPR arrays were detected purely from unassembled metagenomic reads. De novo detection was performed with Crass (Skennerton, Imelfort & Tyson, 2013) with default parameters, which identifies repeat-spacer-repeat patterns in individual reads, uses these as “seeds” to recruit other CRISPR-containing reads, and then assembles these reads into CRISPR arrays. We further clustered Crass spacers using CD-HIT (Li & Godzik, 2006) with a similarity threshold of 0.9 and aligned them against known direct repeats from CRISPRdb (Grissa, Vergnaud & Pourcel, 2007) to determine the associated host organism.…”

Section: Methodsmentioning

confidence: 99%

Determining virus-host interactions and glycerol metabolism profiles in geographically diverse solar salterns with metagenomics

Moller

Liang

2017

PeerJ

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Solar salterns are excellent model ecosystems for studying virus-microbial interactions because of their low microbial diversity, environmental stability, and high viral density. By using the power of CRISPR spacers to link viruses to their prokaryotic hosts, we explored virus-host interactions in geographically diverse salterns. Using taxonomic profiling, we identified hosts such as archaeal Haloquadratum, Halorubrum, and Haloarcula and bacterial Salinibacter, and we found that community composition related to not only salinity but also local environmental dynamics. Characterizing glycerol metabolism genes in these metagenomes suggested Halorubrum and Haloquadratum possess most dihydroxyacetone kinase genes while Salinibacter possesses most glycerol-3-phosphate dehydrogenase genes. Using two different methods, we detected fewer CRISPR spacers in Haloquadratum-dominated compared with Halobacteriaceae-dominated saltern metagenomes. After CRISPR detection, spacers were aligned against haloviral genomes to map virus to host. While most alignments for each saltern metagenome linked viruses to Haloquadratum walsbyi, there were also alignments indicating interactions with the low abundance taxa Haloarcula and Haloferax. Further examination of the dinucleotide and trinucleotide usage differences between paired viruses and their hosts confirmed viruses and hosts had similar nucleotide usage signatures. Detection of cas genes in the salterns supported the possibility of CRISPR activity. Taken together, our studies suggest similar virus-host interactions exist in different solar salterns and that the glycerol metabolism gene dihydroxyacetone kinase is associated with Haloquadratum and Halorubrum.

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“…3), with alternating spacer and repeat units, results in a computationally identifiable sequence signature. Several bioinformatics tools have been developed to identify CRISPR spacers in bacterial genomes (Biswas et al 2013;Bland et al 2007;Edgar and Myers 2005;Grissa et al 2007;Skennerton et al 2013), and spacer sequences have also been collected in publicly accessible databases (Grissa et al 2007;Rousseau et al 2009).…”

Section: Crisprmentioning

confidence: 99%

Metagenomics of microbial and viral life in terrestrial geothermal environments

Strazzulli

Fusco

Cobucci‐Ponzano

et al. 2017

Rev Environ Sci Biotechnol

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Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme-and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.Keywords Hyperthermophiles Á Geothermal environments Á Microbial and viral metagenomics Á CRISPR Á Enzyme discovery BackgroundHot springs populated by extreme thermophiles (T opt : 65-79°C) and hyperthermophiles (T opt [ 80°C) (DeCastro et al. 2016) are very diverse and some of them show combinations of extreme chemo-physical conditions, such as temperature, acidic or alkaline pHs, high pressure, and high concentrations of salts and heavy metals (López-López et al. 2013). As with all studies of environmental microbiology, our understanding of the composition, functional and physiological dynamics, and evolution of extreme-and hyper-thermophilic microbial consortia has lagged A. Strazzulli Á M. Moracci Á P. Contursi

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Crass: identification and reconstruction of CRISPR from unassembled metagenomic data

Cited by 152 publications

References 30 publications

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

Determining virus-host interactions and glycerol metabolism profiles in geographically diverse solar salterns with metagenomics

Metagenomics of microbial and viral life in terrestrial geothermal environments

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