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151.
Kristin Tennessen Evan Andersen Scott Clingenpeel Christian Rinke Derek S Lundberg James Han Jeff L Dangl Natalia Ivanova Tanja Woyke Nikos Kyrpides Amrita Pati 《The ISME journal》2016,10(1):269-272
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.Recent technological advancements have enabled the large-scale sampling of genomes from uncultured microbial taxa, through the high-throughput sequencing of single amplified genomes (SAGs; Rinke et al., 2013; Swan et al., 2013) and assembly and binning of genomes from metagenomes (GMGs; Cuvelier et al., 2010; Sharon and Banfield, 2013). The importance of these products in assessing community structure and function has been established beyond doubt (Kalisky and Quake, 2011). Multiple Displacement Amplification (MDA) and sequencing of single cells has been immensely successful in capturing rare and novel phyla, generating valuable references for phylogenetic anchoring. However, efforts to conduct MDA and sequencing in a high-throughput manner have been heavily impaired by contamination from DNA introduced by the environmental sample, as well as introduced during the MDA or sequencing process (Woyke et al., 2011; Engel et al., 2014; Field et al., 2014). Similarly, metagenome binning and assembly often carries various errors and artifacts depending on the methods used (Nielsen et al., 2014). Even cultured isolate genomes have been shown to lack immunity to contamination with other species (Parks et al., 2014; Mukherjee et al., 2015). As sequencing of these genome product types rapidly increases, contaminant sequences are finding their way into public databases as reference sequences. It is therefore extremely important to define standardized and automated protocols for quality control and decontamination, which would go a long way towards establishing quality standards for all microbial genome product types.Current procedures for decontamination and quality control of genome sequences in single cells and metagenome bins are heavily manual and can consume hours/megabase when performed by expert biologists. Supervised decontamination typically involves homology-based inspection of ribosomal RNA sequences and protein coding genes, as well as visual analysis of k-mer frequency plots and guanine–cytosine content (Clingenpeel, 2015). Manual decontamination is also possible through the software SmashCell (Harrington et al., 2010), which contains a tool for visual identification of contaminants from a self-organizing map and corresponding U-matrix. Another existing software tool, DeconSeq (Schmieder and Edwards, 2011), automatically removes contaminant sequences, however, the contaminant databases are required input. The former lacks automation, whereas the latter requires prior knowledge of contaminants, rendering both applications impractical for high-throughput decontamination.Here, we introduce ProDeGe, the first fully automated computational protocol for decontamination of genomes. ProDeGe uses a combination of homology-based and sequence composition-based approaches to separate contaminant sequences from the target genome draft. It has been pre-calibrated to discard at least 84% of the contaminant sequence, which results in retention of a median 84% of the target sequence. The standalone software is freely available at http://prodege.jgi-psf.org//downloads/src and can be run on any system that has Perl, R (R Core Team, 2014), Prodigal (Hyatt et al., 2010) and NCBI Blast (Camacho et al., 2009) installed. A graphical viewer allowing further exploration of data sets and exporting of contigs accompanies the web application for ProDeGe at http://prodege.jgi-psf.org, which is open to the wider scientific community as a decontamination service (Supplementary Figure S1).The assembly and corresponding NCBI taxonomy of the data set to be decontaminated are required inputs to ProDeGe (Figure 1a). Contigs are annotated with genes following which, eukaryotic contamination is removed based on homology of genes at the nucleotide level using the eukaryotic subset of NCBI''s Nucleotide database as the reference. For detecting prokaryotic contamination, a curated database of reference contigs from the set of high-quality genomes within the Integrated Microbial Genomes (IMG; Markowitz et al., 2014) system is used as the reference. This ensures that errors in public reference databases due to poor quality of sequencing, assembly and annotation do not negatively impact the decontamination process. Contigs determined as belonging to the target organism based on nucleotide level homology to sequences in the above database are defined as ‘Clean'', whereas those aligned to other organisms are defined as ‘Contaminant''. Contigs whose origin cannot be determined based on alignment are classified as ‘Undecided''. Classified clean and contaminated contigs are used to calibrate the separation in the subsequent 5-mer based binning module, which classifies undecided contigs as ‘Clean'' or ‘Contaminant'' using principal components analysis (PCA) of 5-mer frequencies. This parameter can also be specified by the user. When data sets do not have taxonomy deeper than phylum level, or a single confident taxonomic bin cannot be detected using sequence alignment, solely 9-mer based binning is used due to more accurate overall classification. In the absence of a user-defined cutoff, a pre-calibrated cutoff for 80% or more specificity separates the clean contigs from contaminated sequences in the resulting PCA of the 9-mer frequency matrix. Details on ProDeGe''s custom database, evaluation of the performance of the system and exploration of the parameter space to calibrate ProDeGe for a high accurate classification rate are provided in the Supplementary Material.Open in a separate windowFigure 1(a) Schematic overview of the ProDeGe engine. (b) Features of data sets used to validate ProDeGe: SAGs from the Arabidopsis endophyte sequencing project, MDM project, public data sets found in IMG but not sequenced at the JGI, as well as genomes from metagenomes. All the data and results can be found in Supplementary Table S3.The performance of ProDeGe was evaluated using 182 manually screened SAGs (Figure 1b,Supplementary Table S1) from two studies whose data sets are publicly available within the IMG system: genomes of 107 SAGs from an Arabidopsis endophyte sequencing project and 75 SAGs from the Microbial Dark Matter (MDM) project* (only 75/201 SAGs from the MDM project had 1:1 mapping between contigs in the unscreened and the manually screened versions, hence these were used; Rinke et al., 2013). Manual curation of these SAGs demonstrated that the use of ProDeGe prevented 5311 potentially contaminated contigs in these data sets from entering public databases. Figure 2a demonstrates the sensitivity vs specificity plot of ProDeGe results for the above data sets. Most of the data points in Figure 2a cluster in the top right of the box reflecting a median retention of 89% of the clean sequence (sensitivity) and a median rejection of 100% of the sequence of contaminant origin (specificity). In addition, on average, 84% of the bases of a data set are accurately classified. ProDeGe performs best when the target organism has sequenced homologs at the class level or deeper in its high-quality prokaryotic nucleotide reference database. If the target organism''s taxonomy is unknown or not deeper than domain level, or there are few contigs with taxonomic assignments, a target bin cannot be assessed and thus ProDeGe removes contaminant contigs using sequence composition only. The few samples in Figure 2a that demonstrate a higher rate of false positives (lower specificity) and/or reduced sensitivity typically occur when the data set contains few contaminant contigs or ProDeGe incorrectly assumes that the largest bin is the target bin. Some data sets contain a higher proportion of contamination than target sequence and ProDeGe''s performance can suffer under this condition. However, under all other conditions, ProDeGe demonstrates high speed, specificity and sensitivity (Figure 2). In addition, ProDeGe demonstrates better performance in overall classification when nucleotides are considered than when contigs are considered, illustrating that longer contigs are more accurately classified (Supplementary Table S1).Open in a separate windowFigure 2ProDeGe accuracy and performance scatterplots of 182 manually curated single amplified genomes (SAGs), where each symbol represents one SAG data set. (a) Accuracy shown by sensitivity (proportion of bases confirmed ‘Clean'') vs specificity (proportion of bases confirmed ‘Contaminant'') from the Endophyte and Microbial Dark Matter (MDM) data sets. Symbol size reflects input data set size in megabases. Most points cluster in the top right of the plot, showing ProDeGe''s high accuracy. Median and average overall results are shown in Supplementary Table S1. (b) ProDeGe completion time in central processing unit (CPU) core hours for the 182 SAGs. ProDeGe operates successfully at an average rate of 0.30 CPU core hours per megabase of sequence. Principal components analysis (PCA) of a 9-mer frequency matrix costs more computationally than PCA of a 5-mer frequency matrix used with blast-binning. The lack of known taxonomy for the MDM data sets prevents blast-binning, thus showing longer finishing times than the endophyte data sets, which have known taxonomy for use in blast-binning.All SAGs used in the evaluation of ProDeGe were assembled using SPAdes (Bankevich et al., 2012). In-house testing has shown that reads assembled with SPAdes from different strains or even slightly divergent species of the same genera may be combined into the same contig (Personal communications, KT and Robert Bowers). Ideally, the DNA in a well that gets sequenced belongs to a single cell. In the best case, contaminant sequences need to be at least from a different species to be recognized as such by the homology-based screening stage. In the absence of closely related sequenced organisms, contaminant sequences need to be at least from a different genus to be recognized as such by the composition-based screening stage (Supplementary Material). Thus, there is little risk of ProDeGe separating sequences from clonal populations or strains. We have found species- and genus-level contamination in MDA samples to be rare.To evaluate the quality of publicly available uncultured genomes, ProDeGe was used to screen 185 SAGs and 14 GMGs (Figure 1b). Compared with CheckM (Parks et al., 2014), a tool which calculates an estimate of genome sequence contamination using marker genes, ProDeGe generally marks a higher proportion of sequence as ‘Contaminant'' (Supplementary Table S2). This is because ProDeGe has been calibrated to perform at high specificity levels. The command line version of ProDeGe allows users to conduct their own calibration and specify a user-defined distance cutoff. Further, CheckM only outputs the proportion of contamination, but ProDeGe actually labels each contig as ‘Clean'' or ‘Contaminant'' during the process of automated removal.The web application for ProDeGe allows users to export clean and contaminant contigs, examine contig gene calls with their corresponding taxonomies, and discover contig clusters in the first three components of their k-dimensional space. Non-linear approaches for dimensionality reduction of k-mer vectors are gaining popularity (van der Maaten and Hinton, 2008), but we observed no systematic advantage of using t-Distributed Stochastic Neighbor Embedding over PCA (Supplementary Figure S2).ProDeGe is the first step towards establishing a standard for quality control of genomes from both cultured and uncultured microorganisms. It is valuable for preventing the dissemination of contaminated sequence data into public databases, avoiding resulting misleading analyses. The fully automated nature of the pipeline relieves scientists of hours of manual screening, producing reliably clean data sets and enabling the high-throughput screening of data sets for the first time. ProDeGe, therefore, represents a critical component in our toolkit during an era of next-generation DNA sequencing and cultivation-independent microbial genomics. 相似文献
152.
153.
María Belén Federico María Belén Vallerga Analía Radl Natalia Soledad Paviolo José Luis Bocco Marina Di Giorgio Gastón Soria Vanesa Gottifredi 《PLoS genetics》2016,12(1)
Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. 相似文献
154.
Lizhi Wu Sandeep C. Chaudhary Venkatram R. Atigadda Olga V. Belyaeva Steven R. Harville Craig A. Elmets Donald D. Muccio Mohammad Athar Natalia Y. Kedishvili 《PloS one》2016,11(4)
UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA), the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB) irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations. 相似文献
155.
Pragathi B. Shridhar Lance W. Noll Xiaorong Shi Natalia Cernicchiaro David G. Renter J. Bai T. G. Nagaraja 《PloS one》2016,11(3)
Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzxO104 gene, then colonies were tested individually to identify wzxO104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzxO104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzxO104 has been shown to be present in E. coli O8/O9/O9a, wzxO104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdDO8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzxO104 and negative for wbdDO8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar’s test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzxO104 and samples that were positive for wzxO104, but negative for wbdDO8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7. 相似文献
156.
Jagoda Kicielińska Agnieszka Szczygie? Joanna Rossowska Natalia Anger Katarzyna Kempińska Marta ?witalska Marta Kaszowska Joanna Wietrzyk Janusz Boratyński El?bieta Pajtasz-Piasecka 《PloS one》2016,11(2)
Introduction
Polymyxin B (PmB) belongs to the group of cyclic peptide antibiotics, which neutralize the activity of LPS by binding to lipid A. The aim of this study was to analyze the effect of PmB on the biological activity of lipooligosaccharide (LOS E. coli B,rough form of LPS) in vitro and in experimental metastasis models.Results
Cultures of murine macrophage J774A.1 cells and murine bone marrow-derived dendritic cells (BM-DC) stimulated in vitro with LOS and supplemented with PmB demonstrated a decrease in inflammatory cytokine production (IL-6, IL-10, TNF-α) and down-regulation of CD40, CD80, CD86 and MHC class II molecule expression. Additionally, PmB suspended in drinking water was given to the C57BL/6 mice seven or five days prior to the intravenous injection of B16 or LLC cells and intraperitoneal application of LOS. This strategy of PmB administration was continued throughout the duration of the experiments (29 or 21 days). In B16 model, statistically significant decrease in the number of metastases in mice treated with PmB and LOS (p<0.01) was found on the 14th day of the experiments, whereas the most intensive changes in surface-antigen expression and ex vivo production of IL-6, IL-1β and TNF-α by peritoneal cells were observed 7 days earlier. By contrast, antigen expression and ex vivo production of IL-6, IL-10, IFN-γ by splenocytes remained relatively high and stable. Statistically significant decrease in LLC metastases number was observed after the application of LOS (p<0.01) and in the group of mice preconditioned by PmB and subsequently treated with LOS (LOS + PmB, p<0.01).Conclusions
In conclusion, prolonged in vivo application of PmB was not able to neutralize the LOS-induced immune cell activity but its presence in the organism of treated mice was important in modulation of the LOS-mediated response against the development of metastases. 相似文献157.
Jorge Orós Natalia Montesdeoca María Camacho Alberto Arencibia Pascual Calabuig 《PloS one》2016,11(2)
Aims
The aims of this study were to analyze the causes of stranding of 1,860 loggerhead turtles (Caretta caretta) admitted at the Tafira Wildlife Rehabilitation Center in Gran Canaria Island, Spain, from 1998 to 2014, and to analyze the outcomes of the rehabilitation process to allow meaningful auditing of its quality.Methods
Primary causes of morbidity were classified into seven categories: entanglement in fishing gear and/or plastics, ingestion of hooks and monofilament lines, trauma, infectious disease, crude oil, other causes, and unknown/undetermined. Final dispositions were calculated as euthanasia (Er), unassisted mortality (Mr), and release (Rr) rates. Time to death (Td) for euthanized and dead turtles, and length of stay for released (Tr) turtles were evaluated.Results
The most frequent causes of morbidity were entanglement in fishing gear and/or plastics (50.81%), unknown/undetermined (20.37%), and ingestion of hooks (11.88%). The final disposition of the 1,634 loggerhead turtles admitted alive were: Er = 3.37%, Mr = 10.34%, and Rr = 86.29%. Er was significantly higher in the trauma category (18.67%) compared to the other causes of admission. The highest Mr was observed for turtles admitted due to trauma (30.67%). The highest Rr was observed in the crude oil (93.87%) and entanglement (92.38%) categories. The median Tr ranged from 12 days (unknown) to 70 days (trauma).Conclusions
This survey is the first large-scale epidemiological study on causes of stranding and mortality of Eastern Atlantic loggerheads and demonstrates that at least 71.72% of turtles stranded due to anthropogenic causes. The high Rr (86.29%) emphasizes the importance of marine rehabilitation centers for conservation purposes. The stratified analysis by causes of admission of the three final disposition rates, and the parameters Td and Tr should be included in the outcome research of the rehabilitation process of sea turtles in order to allow comparative studies between marine rehabilitation centers around the world. 相似文献158.
Beatriz Martín-Fernández Alfonso Rubio-Navarro Isabel Cortegano Sandra Ballesteros Mario Alía Pablo Cannata-Ortiz Elena Olivares-álvaro Jesús Egido Belén de Andrés María Luisa Gaspar Natalia de las Heras Vicente Lahera Juan Antonio Moreno 《PloS one》2016,11(1)
We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt–treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt–treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation. 相似文献
159.
Joonbae Seo Natalia N. Singh Eric W. Ottesen Senthilkumar Sivanesan Maria Shishimorova Ravindra N. Singh 《PloS one》2016,11(4)
Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein. 相似文献
160.
Natalia J. Martinez Ganesha Rai Adam Yasgar Wendy A. Lea Hongmao Sun Yuhong Wang Diane K. Luci Shyh-Ming Yang Kana Nishihara Shunichi Takeda Mohiuddin Irina Earnshaw Tetsuya Okada Kazutoshi Mori Kelli Wilson Gregory J. Riggins Menghang Xia Maurizio Grimaldi Ajit Jadhav David J. Maloney Anton Simeonov 《PloS one》2016,11(11)