caRpools: an R package for exploratory data analysis and documentation of pooled CRISPR/Cas9 screens.

MOTIVATION: Genetic screens by CRISPR/Cas9-mediated genome engineering have become a powerful tool for functional genomics. However, there is currently a lack of end-to-end software pipelines to analyze CRISPR/Cas9 screens based on next generation sequencing. RESULTS: The CRISPR-AnalyzeR for pooled screens (caRpools) is an R package for exploratory data analysis that provides a complete workflow to analyze CRISPR/Cas9 screens. To further support the analysis of large-scale screens, caRpools integrates screening documentation and generation of standardized analysis reports. AVAILABILITY AND IMPLEMENTATION: caRpools, manuals and an open virtual appliance are available at http://github.com/boutroslab/caRpools.

GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update.

RNA interference (RNAi) represents a powerful method to systematically study loss-of-function phenotypes on a large scale with a wide variety of biological assays, constituting a rich source for the assignment of gene function. The GenomeRNAi database (http://www.genomernai.org) makes available RNAi phenotype data extracted from the literature for human and Drosophila. It also provides RNAi reagent information, along with an assessment as to their efficiency and specificity. This manuscript describes an update of the database previously featured in the NAR Database Issue. The new version has undergone a complete re-design of the user interface, providing an intuitive, flexible framework for additional functionalities. Screen information and gene-reagent-phenotype associations are now available for download. The integration with other resources has been improved by allowing in-links via GenomeRNAi screen IDs, or external gene or reagent identifiers. A distributed annotation system (DAS) server enables the visualization of the phenotypes and reagents in the context of a genome browser. We have added a page listing 'frequent hitters', i.e. genes that show a phenotype in many screens, which might guide on-going RNAi studies. Structured annotation guidelines have been established to facilitate consistent curation, and a submission template for direct submission by data producers is available for download.

E-TALEN: a web tool to design TALENs for genome engineering.

Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future.

GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 update.

The GenomeRNAi database (http://www.genomernai.org/) contains phenotypes from published cell-based RNA interference (RNAi) screens in Drosophila and Homo sapiens. The database connects observed phenotypes with annotations of targeted genes and information about the RNAi reagent used for the perturbation experiment. The availability of phenotypes from Drosophila and human screens also allows for phenotype searches across species. Besides reporting quantitative data from genome-scale screens, the new release of GenomeRNAi also enables reporting of data from microscopy experiments and curated phenotypes from published screens. In addition, the database provides an updated resource of RNAi reagents and their predicted quality that are available for the Drosophila and the human genome. The new version also facilitates the integration with other genomic data sets and contains expression profiling (RNA-Seq) data for several cell lines commonly used in RNAi experiments.

web cellHTS2: a web-application for the analysis of high-throughput screening data.

BACKGROUND: The analysis of high-throughput screening data sets is an expanding field in bioinformatics. High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify relevant phenotypic hits. Large-scale RNAi screens are frequently used to identify novel factors that influence a broad range of cellular processes, including signaling pathway activity, cell proliferation, and host cell infection. Here, we present a web-based application utility for the end-to-end analysis of large cell-based screening experiments by cellHTS2. RESULTS: The software guides the user through the configuration steps that are required for the analysis of single or multi-channel experiments. The web-application provides options for various standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and restored for later re-analysis. The web frontend for the cellHTS2 R/Bioconductor package interacts with it through an R-server implementation that enables highly parallel analysis of screening data sets. web cellHTS2 further provides a file import and configuration module for common file formats. CONCLUSIONS: The implemented web-application facilitates the analysis of high-throughput data sets and provides a user-friendly interface. web cellHTS2 is accessible online at http://web-cellHTS2.dkfz.de. A standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 can be downloaded from the web cellHTS2 page. web cellHTS2 is freely distributed under GPL.

Microbial Communities across Global Marine Basins Show Important Compositional Similarities by Depth.

The environmental surveys following the 2010 Deepwater Horizon (DWH) spill identified a variety of hydrocarbon-degrading microorganisms, and laboratory studies with field-collected water samples then demonstrated faster-than-expected hydrocarbon biodegradation rates at 5°C. Knowledge about microbial community composition, diversity, and functional metabolic capabilities aids in understanding and predicting petroleum biodegradation by microbial communities in situ and is therefore an important component of the petroleum spill response decision-making process. This study investigates the taxonomic composition of microbial communities in six different global basins where petroleum and gas activities occur. Shallow-water communities were strikingly similar across basins, while deep-water communities tended to show subclusters by basin, with communities from the epipelagic, mesopelagic, and bathypelagic zones sometimes appearing within the same cluster. Microbial taxa that were enriched in the water column in the Gulf of Mexico following the DWH spill were found across marine basins. Several hydrocarbon-degrading genera (e.g., Actinobacteria, Pseudomonas, and Rhodobacteriacea) were common across all basins. Other genera such as Pseudoalteromonas and Oleibacter were highly enriched in specific basins.IMPORTANCE Marine microbial communities are a vital component of global carbon cycling, and numerous studies have shown that populations of petroleum-degrading bacteria are ubiquitous in the oceans. Few studies have attempted to distinguish all of the taxa that might contribute to petroleum biodegradation (including, e.g., heterotrophic and nondesignated microbes that respond positively to petroleum and microbes that grow on petroleum as the sole carbon source). This study quantifies the subpopulations of microorganisms that are expected to be involved in petroleum hydrocarbon biodegradation, which is important information during the decision-making process in the event of a petroleum spill accident.

Biodegradation in seawater of PAH and alkylphenols from produced water of a North Sea platform.

Operational planned discharges of produced water (PW) to the marine environment from offshore oil production installations, contain low concentrations of dispersed oil compounds, like polycyclic aromatic hydrocarbons (PAHs) and alkylated phenols (APs). Biotransformation in natural seawater (SW) of naphthalenes/PAHs and phenol/APs in field-collected PW from a North Sea platform was investigated in this biodegradation study. The PW was diluted in SW from a Norwegian fjord, and the biodegradation study was performed in slowly rotating carousels at 13 °C over a period of 62 days. Naphthalenes/PAHs and phenol/APs biotransformation was determined by first-order rate kinetics, after normalization against the recalcitrant biomarker 17α(H),21ß(H)-Hopane. The results from this study showed total biotransformation half-lives ranging from 10 to 19 days for groups of naphthalenes and PAHs, while half-lives for APs (C0- to C9-alkylated) were 10-14 days. Biotransformation half-lives of single compounds ranged from 8 to >100 days for naphthalenes and PAHs (median 16 days), and from 5 to 70 days (median 15 days) for phenols and APs. Four of the tested PAHs (chrysene, benzo(b)fluoranthene, benzo(e)pyrene, benzo(g,h,i)perylene) and one AP (4-tert-butylphenol) showed biotransformation half-lives >50 days. This is one of a few studies that has investigated the potential for biodegradation of PW in natural SW. Methods and data from this study may be used as a part of Risk Based Approaches (RBA) for assessments of environmental fate of PW released to the marine environment and as part of the persistence related to risk.

The Deep-Sea Microbial Community from the Amazonian Basin Associated with Oil Degradation.

One consequence of oil production is the possibility of unplanned accidental oil spills; therefore, it is important to evaluate the potential of indigenous microorganisms (both prokaryotes and eukaryotes) from different oceanic basins to degrade oil. The aim of this study was to characterize the microbial response during the biodegradation process of Brazilian crude oil, both with and without the addition of the dispersant Corexit 9500, using deep-sea water samples from the Amazon equatorial margin basins, Foz do Amazonas and Barreirinhas, in the dark and at low temperatures (4°C). We collected deep-sea samples in the field (about 2570 m below the sea surface), transported the samples back to the laboratory under controlled environmental conditions (5°C in the dark) and subsequently performed two laboratory biodegradation experiments that used metagenomics supported by classical microbiological methods and chemical analysis to elucidate both taxonomic and functional microbial diversity. We also analyzed several physical-chemical and biological parameters related to oil biodegradation. The concomitant depletion of dissolved oxygen levels, oil droplet density characteristic to oil biodegradation, and BTEX concentration with an increase in microbial counts revealed that oil can be degraded by the autochthonous deep-sea microbial communities. Indigenous bacteria (e.g., Alteromonadaceae, Colwelliaceae, and Alcanivoracaceae), archaea (e.g., Halobacteriaceae, Desulfurococcaceae, and Methanobacteriaceae), and eukaryotic microbes (e.g., Microsporidia, Ascomycota, and Basidiomycota) from the Amazonian margin deep-sea water were involved in biodegradation of Brazilian crude oil within less than 48-days in both treatments, with and without dispersant, possibly transforming oil into microbial biomass that may fuel the marine food web.

Biotransformation of potentially persistent alkylphenols in natural seawater.

Produced water (PW) discharged to the marine environment may contain both natural substances and industrial chemicals that are potentially persistent, bioaccumulating and toxic (PBT). Identification of substances as PBT is dependent upon accurate assessment of biodegradation rates, but these measurements can be impeded where substances exhibit inherently low solubility in water. Examples of substances of this kind include some alkylated phenols (APs). Biotransformation of three APs, suspected to be PBT compounds in PW, was investigated by adopting a new methodology in which they were immobilized to hydrophobic adsorbents submerged in natural seawater. These compounds were not ready biodegradable by conventional screening biochemical oxygen demand (BOD) methods at high concentrations (2 mg/L). However, potential biodegradability for two of the three APs were demonstrated by the immobilization method at low concentrations (appr. 100 µg/L), with biotransformation half-lives <50 days. Thus, standard screening tests should be supplemented by biodegradation methods suited for testing of poorly soluble substances before the persistence of potential PBT substances are defined.

Assimilation of toluene carbon along a bacteria-protist food chain determined by 13C-enrichment of biomarker fatty acids.

A food chain consisting of toluene, toluene-degrading Pseudomonas sp. PS+ and a bacterivorous flagellated amoebae Vahlkampfia sp. was established in a batch culture. This culture was amended with [U-13C]toluene and served as a model system to elucidate the flux of carbon in the food chain by quantifying bacterial biovolumes and 13C enrichment of phospholipid fatty acid (PLFA) biomarkers of the bacteria and the heterotrophic protists. Major PLFA detected in the batch co-culture included those derived from Pseudomonas sp. PS+ (16:1omega7c and 18:1omega7c) and Vahlkampfia sp. (20:4omega6c and 20:3omega6c). A numerical model including consumption of toluene by the bacteria and predation of the bacteria by the heterotrophic protists was adjusted to the measured toluene carbon, bacterial carbon and delta13C values of bacterial and protist biomass. Using this model, we estimated that 28+/-7% of the consumed toluene carbon was transformed into bacterial biomass, and 12+/-4% of the predated bacterial carbon was incorporated into heterotrophic protist biomass. Our study showed that the 13C enrichment of PLFA biomarkers coupled to biomass determination via biovolume calculations is a suitable method to trace carbon fluxes in protist-inclusive microbial food chains because it does not require the separation of protist cells from bacterial cells and soil particles.

Aerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials.

The aerobic biodegradability of iso-butanol, a new biofuel, and its impact on benzene, toluene, ethylbenzene and xylenes (BTEX) degradation was investigated in aerobic microcosms consisting of groundwater and sediment from a California site with a history of gasoline contamination. To the best of our knowledge this is the first study directly examining the effects of iso-butanol on BTEX degradation. Microcosms that received either low (68 µM) or high (3400 µM) concentrations of iso-butanol showed complete biodegradation of iso-butanol within 7 and 23 d, respectively, of incubation at 15°C under aerobic conditions. A maximum utilization rate coefficient of 2.3±0.1×10⁻7 µmol cell⁻¹ h⁻¹ and a half saturation constant of 610±54 µM were regressed from the iso-butanol data. Iso-butanol biodegradation resulted in transient formation of the degradation intermediate products iso-butylaldehyde and iso-butyric acid, and both compounds were subsequently degraded within the timeframe of the experiments. Ethanol was biodegraded more slowly than iso-butanol. Ethanol also exhibited greater adverse impacts on BTEX biodegradation than iso-butanol. Results of the study suggest that iso-butanol added to fuels will be readily biodegraded in the environment under aerobic conditions without the accumulation of major intermediate products (iso-butylaldehyde and iso-butyric acid), and that it will pose less impacts on BTEX biodegradation than ethanol.

Anaerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials.

Biologically produced iso-butanol is currently being considered as an additive in gasoline blends. To evaluate its potential environmental fate in groundwater aquifers, a laboratory microcosm study was performed to evaluate iso-butanol biodegradation under various anaerobic conditions (nitrate-reducing, sulfate-reducing and methanogenic). The impacts of iso-butanol on benzene, toluene, ethylbenzene, and total xylenes (BTEX) biodegradation were also assessed, and microcosms prepared using ethanol instead of iso-butanol were evaluated to provide a basis for comparison. Iso-butanol was biodegraded under all conditions studied, with an observed apparent first-order rate constant ranging from approximately 0.2 d⁻¹ (nitrate-reducing) to approximately 0.02 d⁻¹ (sulfate-reducing). Iso-butanol typically was degraded in a time frame that was shorter than or similar to BTEX compounds. Iso-butyric acid and trace levels of iso-butylaldehyde were identified as transient intermediates, and both of these compounds were subsequently degraded within the time frame of the experiments. Iso-butanol and ethanol were biodegraded in similar time frames under methanogenic conditions. Under sulfate-reducing conditions, iso-butanol biodegradation initially proceeded more slowly than ethanol, and then increased to a rate greater than that observed for ethanol; this observation likely was due to the growth of iso-butanol degrading bacteria. Iso-butanol generally exhibited less adverse impacts on BTEX biodegradations than ethanol under the anaerobic conditions studied. In some cases, addition of iso-butanol enhanced the rate of TEX biodegradation.

Duplex polymerase chain reaction quantification of human cells in a murine background.

Studies of the regenerative potential of human stem cells commonly involve their transplantation into immune-deficient mice or in vitro coculture with mouse cells. The optimal use of such models requires the detection and quantification of relatively low numbers of human cells in a murine background. We report here a duplex polymerase chain reaction (PCR) approach involving the coamplification of human-and mouse-specific repetitive sequences. The determination of product ratios compensates against variations in sample quality and enables quantitation from >50% down to 0.01% human-in-mouse from a single reaction. Product ratios are determined by standard electrophoresis of end-stage PCR reactions followed by image analysis techniques using freely available software, with no requirement for real-time PCR. The approach has been used to analyze tissue from mice transplanted with human cells and cocultures between differentiating mouse embryonal stem cells and human umbilical cord blood cells.

Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms.

Abstract Microbial sulfate reduction is an important metabolic activity in many reduced habitats. However, little is known about the sulfate-reducing communities inhabiting petroleum hydrocarbon (PHC)-contaminated freshwater aquifer sediments. The purpose of this study was to identify the groups of sulfate-reducing bacteria (SRB) selectively stimulated when sediment from a PHC-contaminated freshwater aquifer was incubated in sulfate-reducing aquifer microcosms that were amended with specific carbon sources (acetate, butyrate, propionate, lactate, and citrate). After 2 months of incubation, the SRB community was characterized using phospholipid fatty acid (PLFA) analysis combined with multivariate statistics as well as fluorescence in situ hybridization (FISH). Molybdate was used to specifically inhibit SRB in separate microcosms to investigate the contribution of non-SRB to carbon source degradation. Results indicated that sulfate reduction in the original sediment was an important process but was limited by the availability of sulfate. Substantially lower amounts of acetate and butyrate were degraded in molybdate treatments as compared to treatments without molybdate, suggesting that SRB were the major bacterial group responsible for carbon source turnover in microcosms. All of the added carbon sources induced changes in the SRB community structure. Members of the genus Desulfobulbus were present but not active in the original sediment but an increase of the fatty acids 15:1omega6c and 17:1omega6c and FISH results showed an enrichment of these bacteria in microcosms amended with propionate or lactate. The appearance of cy17:0 revealed that bacteria affiliated with the Desulfobacteriaceae were responsible for acetate degradation. Desulfovibrio and Desulfotomaculum spp. were not important populations within the SRB community in microcosms because they did not proliferate on carbon sources usually favored by these organisms. Metabolic, PLFA, and FISH results provided information on the SRB community in a PHC-contaminated freshwater environment, which exhibited stimulation patterns similar to other (e.g. marine) environments.

Field-scale C-labeling of phospholipid fatty acids (PLFA) and dissolved inorganic carbon: tracing acetate assimilation and mineralization in a petroleum hydrocarbon-contaminated aquifer.

This study was conducted to determine the feasibility of labeling phospholipid-derived fatty acids (PLFA) of an active microbial population with a (13)C-labeled organic substrate in the denitrifying zone of a petroleum hydrocarbon-contaminated aquifer during a single-well push-pull test. Anoxic test solution was prepared from 500 l of groundwater with addition of 0.5 mM Br(-) as a conservative tracer, 0.5 mM NO(3) (-), and 0.25 mM [2-(13)C]acetate. At 4, 23 and 46 h after injection, 1000 l of test solution/groundwater mixture were sequentially extracted. During injection and extraction phases we measured Br(-), NO(3) (-) and acetate concentrations, characterized the microbial community structure by PLFA and fluorescent in situ hybridization (FISH) analyses, and determined (13)C/(12)C ratios in dissolved inorganic carbon (DIC) and PLFA. Computed first-order rate coefficients were 0.63+/-0.08 day(-1) for NO(3) (-) and 0.70+/-0.05 day(-1) for acetate consumption. Significant (13)C incorporation in DIC and PLFA was detected as early as 4 h after injection. At 46 h we measured delta(13)C values of up to 5614 per thousand in certain PLFA (especially monounsaturated fatty acids), and up to 59.8 per thousand in extracted DIC. Profiles of enriched PLFA and FISH analysis suggested the presence of active denitrifiers. Our results demonstrate the applicability of (13)C labeling of PLFA and DIC in combination with FISH to link microbial structure and activities at the field scale during a push-pull test.

Characterization of multiple-substrate utilization by anthracene-degrading Mycobacterium frederiksbergense LB501T.

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by Mycobacterium frederiksbergense LB501T under various substrate regimens. The distinct (13)C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that M. frederiksbergense LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of M. frederiksbergense LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by M. frederiksbergense LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.

Influence of the growth substrate on ester-linked phospho- and glycolipid fatty acids of PAH-degrading Mycobacterium sp. LB501T.

The influences of poorly water-soluble anthracene on ester-linked phospholipid fatty acid (PLFA) and glycolipid fatty acid (GLFA) profiles of Mycobacterium sp. LB501T were studied. Bacteria were cultivated on either anthracene or glucose (one culture with successively amended small doses of this substrate and one with excess concentrations) to distinguish between influences of the chemical structure and the bioavailability of the growth substrate. Results revealed that GLFA and PLFA profiles of M. sp. LB501T depended on the availability and the structure of the carbon source. Fatty acid profiles obtained with anthracene differed from those obtained with excess glucose. They were interpreted as a specific adaptation to this poorly bioavailable polycyclic aromatic hydrocarbon (PAH). In contrast, profiles obtained with low glucose concentrations showed clear signs of starvation stress. Stable carbon isotopic ratios (delta13C) of GLFA and PLFA of M. sp. LB501T were analysed to characterize the 13C-fractionation during the biosynthesis of individual fatty acids and to evaluate their value as markers for substrate usage. Although the delta13C values of PLFA and GLFA showed differential isotope fractionation during anthracene- and glucose-degradation, they were sufficiently distinct to be used as signatures of bacterial substrate usage.