ProDeGe: a computational protocol for fully automated decontamination of genomes

Single amplified genomes and genomes assembled from metagenomes have enabled the exploration of uncultured microorganisms at an unprecedented scale. However, both these types of products are plagued by contamination. Since these genomes are now being generated in a high-throughput manner and sequences from them are propagating into public databases to drive novel scientific discoveries, rigorous quality controls and decontamination protocols are urgently needed. Here, we present ProDeGe (Protocol for fully automated Decontamination of Genomes), the first computational protocol for fully automated decontamination of draft genomes. ProDeGe classifies sequences into two classes—clean and contaminant—using a combination of homology and feature-based methodologies. On average, 84% of sequence from the non-target organism is removed from the data set (specificity) and 84% of the sequence from the target organism is retained (sensitivity). The procedure operates successfully at a rate of ~0.30 CPU core hours per megabase of sequence and can be applied to any type of genome sequence

Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells

Genomic features of bacterial adaptation to plants

Author(s): Levy, A; Salas Gonzalez, I; Mittelviefhaus, M; Clingenpeel, S; Herrera Paredes, S; Miao, J; Wang, K; Devescovi, G; Stillman, K; Monteiro, F; Rangel Alvarez, B; Lundberg, DS; Lu, TY; Lebeis, S; Jin, Z; McDonald, M; Klein, AP; Feltcher, ME; Rio, TG; Grant, SR; Doty, SL; Ley, RE; Zhao, B; Venturi, V; Pelletier, DA; Vorholt, JA; Tringe, SG; Woyke, T; Dangl, JL | Abstract: © 2017 The Author(s). Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering

Cloning and In Situ Expression Studies of the Hydrogenobaculum Arsenite Oxidase Genes▿

Novel arsenite [As(III)] oxidase structural genes (aoxAB) were cloned from Hydrogenobaculum bacteria isolated from an acidic geothermal spring. Reverse transcriptase PCR demonstrated expression throughout the outflow channel, and the aoxB cDNA clones exhibited distribution patterns relative to the physicochemical gradients in the spring. Microelectrode analyses provided evidence of quantitative As(III) transformation within the microbial mat

UV Decontamination of MDA Reagents for Single Cell Genomics

Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we address a key challenge inherent to this approach and propose a solution for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a single cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV irradiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Consequently, we also find that increased UV exposure to Phi29 does not adversely affect genome coverage of MDA amplified single cells. While additional challenges in single cell genomics remain to be resolved, the proposed methodology is relatively quick and simple and we believe that its application will be of high value for future single cell sequencing projects

Helmets for snow sports: Prevalence, trends, predictors and attitudes to use

Background: Traumatic head injury is the leading cause of mortality and serious morbidity in snow sports and is increasing in incidence. Helmet use in snow sports can reduce the incidence of head injury by up to 60%. Mandatory helmet use is not legislated in most recreational settings for snow sports. This study aimed to quantify the prevalence and trends of helmet use by skiers and snowboarders as well as to investigate predictors of helmet use and reasons why they are worn. Methods: A cross-sectional study including observation of skiers and snowboarders allocated to adult and child groups (5,267 persons), retrospective review of ski patrol accident report forms recorded between 2003 and 2008 (3,984), and completion of specially designed questionnaires by randomly approached snow sport participants (1,029). Results: In 2008, 16% of adults and 67% of children wore helmets. Helmet use increased for adult and child participants between 2003 and 2008. Children, men, and snowboarders were significantly more likely to wear a helmet than their respective counterparts. Significant predictors of helmet use were level of experience, past major crash requiring medical assistance, snow sport lessons, and location of activity. Key reasons for helmet use and disuse were identified. Discussion: Child helmet use far exceeded adult helmet use. Rates of voluntary helmet use are increasing but many remain unprotected from the benefits of a helmet. Identification of the snow sport participants least likely to wear a helmet and their reasons for not doing so should allow targeted helmet promotion and injury prevention strategies to reduce serious head injuries in snow sports.Thomas P. Cundy, Brenton J. Systermans, William J. Cundy, Peter J. Cundy, Nancy E. Briggs, and Jeffrey B. Robinso

Phylogenomics suggests oxygen availability as a driving force in Thaumarchaeota evolution

Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in marine and terrestrial habitats, playing a major role in the global nitrogen cycle. However, their evolutionary history remains unexplored, which limits our understanding of their adaptation mechanisms. Here, our comprehensive phylogenomic tree of Thaumarchaeota supports three sequential events: origin of AOA from terrestrial non-AOA ancestors, colonization of the shallow ocean, and expansion to the deep ocean. Careful molecular dating suggests that these events coincided with the Great Oxygenation Event around 2300 million years ago (Mya), and oxygenation of the shallow and deep ocean around 800 and 635-560 Mya, respectively. The first transition was likely enabled by the gain of an aerobic pathway for energy production by ammonia oxidation and biosynthetic pathways for cobalamin and biotin that act as cofactors in aerobic metabolism. The first transition was also accompanied by the loss of dissimilatory nitrate and sulfate reduction, loss of oxygen-sensitive pyruvate oxidoreductase, which reduces pyruvate to acetyl-CoA, and loss of the Wood-Ljungdahl pathway for anaerobic carbon fixation. The second transition involved gain of a K+ transporter and of the biosynthetic pathway for ectoine, which may function as an osmoprotectant. The third transition was accompanied by the loss of the uvr system for repairing ultraviolet light-induced DNA lesions. We conclude that oxygen availability drove the terrestrial origin of AOA and their expansion to the photic and dark oceans, and that the stressors encountered during these events were partially overcome by gene acquisitions from Euryarchaeota and Bacteria, among other sources

Surface Reaction Models of Protocells

Single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we show that compared to cryopreservation, lower quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencin

Assembling Single-Cell Genomes and Mini-Metagenomes From Chimeric MDA Products

Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We present algorithms for identifying chimeric edges and resolving complex bulges in de Bruijn graphs, which significantly improve single-cell assemblies. We further describe applications of the single-cell assembler SPAdes to a new approach for capturing and sequencing “microbial dark matter” that forms small pools of randomly selected single cells (called a mini-metagenome) and further sequences all genomes from the mini-metagenome at once. On single-cell bacterial datasets, SPAdes improves on the recently developed E+V-SC and IDBA-UD assemblers specifically designed for single-cell sequencing. For standard (cultivated monostrain) datasets, SPAdes also improves on A5, ABySS, CLC, EULER-SR, Ray, SOAPdenovo, and Velvet. Thus, recently developed single-cell assemblers not only enable single-cell sequencing, but also improve on conventional assemblers on their own turf. SPAdes is available for free online download under a GPLv2 license