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.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.     

Preclinical pharmacokinetics of MHAA4549A,a human monoclonal antibody to influenza A virus,and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ～2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.     

Interaction of Catechu Dye with DNA: Spectroscopic and In Silico Approach

Catechin, a yellow colored molecule obtained from the wood of Acacia catechu was analyzed for its interaction with synthetic DNA duplexes using spectroscopic analysis. UV-Visible spectroscopic analysis revealed the non-intercalative binding mode. Fourier Transform Infrared spectroscopy (FTIR) analysis expose chemical shift indicated by various vibrational stretches and an increase in the intensity of base stacking was observed by Circular Dichroism (CD), respectively. This inference was further confirmed through nuclear staining technique and also in electrophoretic technique; the dye quenches the fluorescent intensity of ethidium bromide. The result of fluorescence spectroscopy was in concordance with the electrophoretic technique. In addition, the spectroscopic results were in accordance with the molecular docking studies of specific catechin compound from the catechu dye with CT-DNA. This kind of site specificity is a gain in the medicinal field as the drug can be DNA targeted for cancer therapeutics. The present work reveals that catechu dye has a noteworthy application in the field of medical bioscience.     

Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of k_cat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.     

Caenorhabditis elegans mutants resistant to attachment of Yersinia biofilms

The detailed composition and structure of the Caenorhabditis elegans surface are unknown. Previous genetic studies used antibody or lectin binding to identify srf genes that play roles in surface determination. Infection by Microbacterium nematophilum identified bus (bacterially unswollen) genes that also affect surface characteristics. We report that biofilms produced by Yersinia pestis and Y. pseudotuberculosis, which bind the C. elegans surface predominantly on the head, can be used to identify additional surface-determining genes. A screen for C. elegans mutants with a biofilm absent on the head (Bah) phenotype identified three novel genes: bah-1, bah-2, and bah-3. The bah-1 and bah-2 mutants have slightly fragile cuticles but are neither Srf nor Bus, suggesting that they are specific for surface components involved in biofilm attachment. A bah-3 mutant has normal cuticle integrity, but shows a stage-specific Srf phenotype. The screen produced alleles of five known surface genes: srf-2, srf-3, bus-4, bus-12, and bus-17. For the X-linked bus-17, a paternal effect was observed in biofilm assays.     

Long-Range Surface Plasmon Resonance–Based Sensitivity Study on D-shaped Ag-MgF2-Coated Models with Analyte Variations

In this report, a novel D-shaped long-range surface plasmon resonance (LRSPR) fiber base sensor has been introduced. The demonstration of proposed sensor involves two D-shaped silver-coated models to study the sensitivity responses. The entire study with the constructed models is based on a single-mode fiber. The models are multilayered consisting of metal, dielectric, and analyte as separate layers. Silver (Ag) and magnesium fluoride (MgF2) strips are used as metal and dielectric layers respectively. The constituency of analyte as an interface excellently standardized the models for sensitivity detection. In this report, a large range of analyte refractive indices (RI) which varies from 1.33 to 1.38 is appraised for the proposed models to characterize the sensitivity. The entire context is encompassed by the wavelength region from 450 to 850 nm with an interval of 20 nm. Sensitivities in this report are measured based on the analyte position from the core and metal for both models. For each of the two models, the analyte is placed as the top layer. RIs of the applied metal (Ag) are measured using the Drude-Lorentz formula. The simulated sensitivities for model-1 and model-2 vary from 6.3?×?10³ nm/RIU to 8.7?×?10³ nm/RIU.

     

Complete genome sequence of the orange-red pigmented, radioresistant Deinococcus proteolyticus type strain (MRP(T))

Deinococcus proteolyticus (ex Kobatake et al. 1973) Brook and Murray 1981 is one of currently 47 species in the genus Deinococcus within the family Deinococcaceae. Strain MRP(T) was isolated from feces of Lama glama and possesses extreme radiation resistance, a trait is shares with various other species of the genus Deinococcus, with D. proteolyticus being resistant up to 1.5 Mrad of gamma radiation. Strain MRP(T) is of further interest for its carotenoid pigment. The genome presented here is only the fifth completed genome sequence of a member of the genus Deinococcus (and the forth type strain) to be published, and will hopefully contribute to a better understanding of how members of this genus adapted to high gamma- or UV ionizing-radiation. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,886,836 bp long genome with its four large plasmids of lengths 97 kbp, 132 kbp, 196 kbp and 315 kbp harbors 2,741 protein-coding and 58 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.