Towards comprehensive understanding of bacterial genetic diversity: large-scale amplifications in Bordetella pertussis and Mycobacterium tuberculosis.

Bacterial genetic diversity is often described solely using base-pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplication/amplifications are thought to be widespread among bacteria but due to their often-intractable size and instability, comprehensive studies of these mutations are rare. We define a methodology to investigate amplifications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for amplifications to occur. Analysis of data for 2430 B. pertussis isolates identified 272 putative amplifications, of which 94 % were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of amplifications, suggesting unstable and sporadic characteristics. Genome instability was further described in vitro using long-read sequencing via the Nanopore platform, which revealed that clonally derived laboratory cultures produced heterogenous populations rapidly. We extended this research to analyse a population of 1000 isolates of another important pathogen, Mycobacterium tuberculosis. We found 590 amplifications in M. tuberculosis, and like B. pertussis, these occurred primarily at hotspots. Genes amplified in B. pertussis include those involved in motility and respiration, whilst in M. tuberuclosis, functions included intracellular growth and regulation of virulence. Using publicly available short-read data we predicted previously unrecognized, large amplifications in B. pertussis and M. tuberculosis. This reveals the unrecognized and dynamic genetic diversity of B. pertussis and M. tuberculosis, highlighting the need for a more holistic understanding of bacterial genetics.

Characterization of Lysinibacillus fusiformis strain S4C11: In vitro, in planta, and in silico analyses reveal a plant-beneficial microbe.

Despite sharing many of the traits that have allowed the genus Bacillus to gain recognition for its agricultural relevance, the genus Lysinibacillus is not as well-known and studied. The present study employs in vitro, in vivo, in planta, and in silico approaches to characterize Lysinibacillus fusiformis strain S4C11, isolated from the roots of an apple tree in northern Italy. The in vitro and in vivo assays demonstrated that strain S4C11 possesses an antifungal activity against different fungal pathogens, and is capable of interfering with the germination of Botrytis cinerea conidia, as well as of inhibiting its growth through the production of volatile organic molecules. In planta assays showed that the strain possesses the ability to promote plant growth, that is not host-specific, both in controlled conditions and in a commercial nursery. Biocontrol assays carried out against phytopathogenic viruses gave contrasting results, suggesting that the strain does not activate the host's defense pathways. The in silico analyses were carried out by sequencing the genome of the strain through an innovative approach that combines Illumina and High-Definition Mapping methods, allowing the reconstruction of a main chromosome and two plasmids from strain S4C11. The analysis of the genes encoded by the genome contributed to the characterization of the strain, detecting genes related to the biocontrol effect detected in the experimental trials.

Author Correction: A robust benchmark for detection of germline large deletions and insertions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A robust benchmark for detection of germline large deletions and insertions.

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution and comprehensiveness. To help translate these methods to routine research and clinical practice, we developed a sequence-resolved benchmark set for identification of both false-negative and false-positive germline large insertions and deletions. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle Consortium integrated 19 sequence-resolved variant calling methods from diverse technologies. The final benchmark set contains 12,745 isolated, sequence-resolved insertion (7,281) and deletion (5,464) calls ≥50 base pairs (bp). The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.51 Gbp and 5,262 insertions and 4,095 deletions supported by ≥1 diploid assembly. We demonstrate that the benchmark set reliably identifies false negatives and false positives in high-quality SV callsets from short-, linked- and long-read sequencing and optical mapping.

Anthropogenic disturbance and biodiversity of marine benthic communities in Antarctica: a regional comparison.

The impacts of two Antarctic stations in different regions, on marine sediment macrofaunal communities were compared: McMurdo, a very large station in the Ross Sea; and Casey, a more typical small station in East Antarctica. Community structure and diversity were compared along a gradient of anthropogenic disturbance from heavily contaminated to uncontaminated locations. We examined some of the inherent problems in comparing data from unrelated studies, such as different sampling methods, spatial and temporal scales of sampling and taxonomic uncertainty. These issues generated specific biases which were taken into account when interpreting patterns. Control sites in the two regions had very different communities but both were dominated by crustaceans. Community responses to anthropogenic disturbance (sediment contamination by metals, oils and sewage) were also different. At McMurdo the proportion of crustaceans decreased in disturbed areas and polychaetes became dominant, whereas at Casey, crustaceans increased in response to disturbance, largely through an increase in amphipods. Despite differing overall community responses there were some common elements. Ostracods, cumaceans and echinoderms were sensitive to disturbance in both regions. Capitellid, dorvelleid and orbiniid polychaetes were indicative of disturbed sites. Amphipods, isopods and tanaids had different responses at each station. Biodiversity and taxonomic distinctness were significantly lower at disturbed locations in both regions. The size of the impact, however, was not related to the level of contamination, with a larger reduction in biodiversity at Casey, the smaller, less polluted station. The impacts of small stations, with low to moderate levels of contamination, can thus be as great as those of large or heavily contaminated stations. Regional broad scale environmental influences may be important in determining the composition of communities and thus their response to disturbance, but there are some generalizations regarding responses which will aid future management of stations.

Recruitment, growth and mortality of an Antarctic hexactinellid sponge, Anoxycalyx joubini.

Polar ecosystems are sensitive to climate forcing, and we often lack baselines to evaluate changes. Here we report a nearly 50-year study in which a sudden shift in the population dynamics of an ecologically important, structure-forming hexactinellid sponge, Anoxycalyx joubini was observed. This is the largest Antarctic sponge, with individuals growing over two meters tall. In order to investigate life history characteristics of Antarctic marine invertebrates, artificial substrata were deployed at a number of sites in the southern portion of the Ross Sea between 1967 and 1975. Over a 22-year period, no growth or settlement was recorded for A. joubini on these substrata; however, in 2004 and 2010, A. joubini was observed to have settled and grown to large sizes on some but not all artificial substrata. This single settlement and growth event correlates with a region-wide shift in phytoplankton productivity driven by the calving of a massive iceberg. We also report almost complete mortality of large sponges followed over 40 years. Given our warming global climate, similar system-wide changes are expected in the future.

Mapping and sequencing DNA using nanopores and nanodetectors.

Even prior to the introduction of capillary DNA sequencers, nanopores were discussed as a low-cost, high-throughput substrate for sequencing. Since then, other next-generation sequencing technologies have been developed and achieved widespread use, but nanopores have lagged behind due to difficulties in generating usable sequence data. The practical and theoretical issues of translocation speed and signal detection encountered when attempting to sequence DNA with nanopores are discussed. Various methods that different laboratories have used to overcome difficulties in biologically based and solid-state nanopores are also presented. Different approaches designed to circumvent the overriding issue of detecting signals from individual bases in a time-resolved manner in nanopores are described. For example, genomic positional sequencing utilizes hybridization of short oligonucleotide probes to very long DNA templates and then detects these probes by variations in current blockade in solid-state nanodetectors. The positions of the probes relative to each other and relative to the ends of the DNA are determined by measuring the time between current blockade peaks. By assembling many such measurements, it is possible to overcome the problems encountered when attempting to sequence DNA at high speed in nanopores, providing the potential for true de novo sequencing of large genomes on a routine basis.

Effect of sample area and sieve size on benthic macrofaunal community condition assessments in California enclosed bays and estuaries.

Benthic macrofauna are used extensively for environmental assessment, but the area sampled and sieve sizes used to capture animals often differ among studies. Here, we sampled 80 sites using 3 different sized sampling areas (0.1, 0.05, 0.0071 m(2)) and sieved those sediments through each of 2 screen sizes (0.5, 1 mm) to evaluate their effect on number of individuals, number of species, dominance, nonmetric multidimensional scaling (MDS) ordination, and benthic community condition indices that are used to assess sediment quality in California. Sample area had little effect on abundance but substantially affected numbers of species, which are not easily scaled to a standard area. Sieve size had a substantial effect on both measures, with the 1-mm screen capturing only 74% of the species and 68% of the individuals collected in the 0.5-mm screen. These differences, though, had little effect on the ability to differentiate samples along gradients in ordination space. Benthic indices generally ranked sample condition in the same order regardless of gear, although the absolute scoring of condition was affected by gear type. The largest differences in condition assessment were observed for the 0.0071-m(2) gear. Benthic indices based on numbers of species were more affected than those based on relative abundance, primarily because we were unable to scale species number to a common area as we did for abundance.

The potential and challenges of nanopore sequencing.

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.

Simultaneous detection and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates in hair by LC-ESI-MS-MS using a single extraction method.

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates from hair using a single extraction method. As part of the method development, Gemini C18, Synergi Hydro RP, and Zorbax Stablebond-Phenyl LC columns were tested with three different mobile phases. Analyte recovery and limit of detection were evaluated for two different solid-phase extraction methods that used Bond Elut Certify and Clean Screen cartridges. Phosphate buffer (pH 5.0) was chosen as the optimum hair incubation medium because of the high stability of cocaine and 6-monoacetylmorphine using this method and faster sample preparation. The optimized method was fully validated. Linearity was established over the concentration range 0.2-10 ng/mg hair, and the correlation coefficients were all greater than 0.99. Total extraction recoveries were greater than 76%, detection limits were between 0.02 and 0.09 ng/mg, and the intra- and interday imprecisions were generally less than 20% in spiked hair. The intra- and interbatch imprecision of the method for a pooled authentic hair sample ranged from 1.4 to 23.4% relative standard deviation (RSD) and 8.3 to 25.4% RSD, respectively, for representative analytes from the different drug groups. The percent matrix effect ranged from 63.5 to 135.6%, with most analytes demonstrating ion suppression. Sixteen postmortem samples collected from suspected drug-related deaths were analyzed for the 17 drugs of abuse and metabolites included in the method. The method was sufficiently sensitive and specific for the analysis of drugs and metabolites in postmortem hair samples. There is scope for the inclusion of other target drugs and metabolites in the method.

Detection of benzodiazepines in hair using ELISA and LC-ESI-MS-MS.

This study was designed to validate an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of nine benzodiazepines in hair. Sixteen hair case samples were tested from drug-related deaths where a positive benzodiazepine blood result was obtained. The case samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h and then neutralized with dibasic phosphate buffer. They were diluted 1:5 with phosphate buffer saline (PBS) prior to analysis. For LC-MS-MS, the samples were incubated overnight in methanol/25% ammonium hydroxide (20:1). The benzodiazepines were extracted by solid phase. Thirteen samples were confirmed positive by LC-MS-MS. The benzodiazepines detected included diazepam, nordiazepam, temazepam, oxazepam, nitrazepam, and lorazepam. Using a cut-off concentration of 0.1 ng/mg oxazepam, the Immunalysis Benzodiazepine Microplate ELISA demonstrated a sensitivity and specificity of 100% and 81%, respectively, compared with LC-MS-MS results.

Validation of the immunalysis microplate ELISA for the detection of methamphetamine in hair.

The object of this study was to validate the Immunalysis Methamphetamine Microplate ELISA for detecting methamphetamine in hair. Twenty-nine scalp hair samples were obtained as routine cases submitted to the National Institute of Scientific Investigation in Seoul by the police. The hair samples were washed with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. The samples were screened using the Immunalysis Methamphetamine Microplate ELISA and confirmed using gas chromatography-mass spectrometry (GC-MS). Twenty-eight hair samples were screened and confirmed as positive for methamphetamine. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h at 60 degrees C. For GC-MS, the samples were extracted for 20 h in methanol containing 1% hydrochloric acid. The methanol/acid solution was evaporated to dryness and the resulting residue was derivatized with trifluoroacetic anhydride. Methamphetamine and amphetamine were detected using selective ion monitoring (SIM) mode. The Immunalysis Methamphetamine Microplate ELISA demonstrated a sensitivity and specificity of 97% and 100%, respectively, using a cut-off concentration of 0.5 ng/mg d-methamphetamine. The ELISA kit showed 63% cross-reactivity with d,l-methamphetamine and did not cross-react to any significant extent with the licit l-methamphetamine isomer. The intra- and interassay precisions were 2.5% and 3.7%, respectively.

Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine.

The purpose of this study was to validate the Immunalysis Buprenorphine Microplate enzyme-linked immunosorbent assay (ELISA) for the detection of buprenorphine in urine samples. Sixty-nine urine samples were obtained from volunteers on the Subutex treatment program and from routine samples submitted to the laboratory for buprenorphine testing. For ELISA analysis, samples were diluted 1:10 with K(2)HPO(4) (0.1M, pH 7.0). The limit of detection was calculated as 0.5 ng/mL buprenorphine. The intra-assay and interday precision was 3.8% (n = 10) and 8.6% (n = 50) respectively at 1 ng/mL buprenorphine. At a low concentration of norbuprenorphine (1 ng/mL), the immunoassay demonstrated a cross-reactivity of 78%. A higher cross-reactivity of 116% was observed at a higher concentration of norbuprenorphine (10 ng/mL). Dihydrocodeine, codeine, tramadol, morphine, propoxyphene, methadone, and EDDP were tested at concentrations of 10 ng/mL and 10,000 ng/mL and demonstrated no cross-reactivity with the assay. For liquid chromatography-tandem mass spectrometry (LC-MS-MS), deuterated internal standard mixture, 1M acetate buffer (pH 5.0), and b-glucuronidase were added to the standards and samples, which were then incubated for 3 h at 60 degrees C. After incubation, 3 mL K(2)HPO(4) (0.1M, pH 6.0) was added and the pH altered to pH 6.0 using 1M KOH. Buprenorphine and norbuprenorphine were subsequently extracted by solid-phase. Twenty-one samples were confirmed positive and 48 samples were confirmed negative by LC-MS-MS. Using a cut-off value of 0.5 ng/mL buprenorphine, the immunoassay demonstrated a sensitivity and specificity of 100%.

DNA sequencing by hybridization using semi-degenerate bases.

One way to enhance the performance of hybridization microarrrays for DNA de novo sequencing is the use of probing patterns with gaps of unsampled positions. Ideally, such gaps could be realized by the inclusion into microarray oligos (probes) of wild-card compounds, referred to as universal bases (which bind nonspecifically to natural bases). The suggested alternative is to deploy in the gap positions degenerate bases, i.e., uniform mixtures of the four natural bases, with ensuing deterioration of the hybridization signal. In this paper, we show that such signal loss is a minor shortcoming, compared with the fact that degenerate bases cannot be treated as universal. Indeed, the substantial spread of hybridization energies at any microarray feature is such that on overwhelming number of mismatches bind more strongly than legal matches. We observed, however, that much narrower energy spreads are exhibited by pairs of bases in the same strength class (A-T and C-G). We call semi-degenerate a gap position realized with bases in the same energy class and show that well-known sequence reconstruction algorithms can be modified to achieve substantial improvements in sequencing effectiveness. For example, with a 4(9)-feature microarray and an acceptable weakening of the hybridization signal, one may achieve lengths of about 4,000 bases (compared with < 250 of the standard uniform method). Our approach also incorporates the use of a spectrum expressed in terms of observed feature melting temperatures (analog spectrum), rather than binary decisions made directly at the biochemical level (digital spectrum). While universal bases represent the ultimate goal of sequencing by hybridization, semidegenerate natural bases are the most effective known substitute.

Benthic changes during 10 years of organic enrichment by McMurdo Station, Antarctica.

A benthic habitat along the coast of McMurdo Station in the Ross Sea, Antarctica is enriched by sewage from the station and altered by hydrocarbons and heavy metals in an adjacent historic dumpsite. We report on 10 years of change in the benthic communities from 1988 to 1998 and compare enrichment effects at Australia's Casey Station, East Antarctica. Despite being 14 km apart, reference communities upcurrent and downcurrent of McMurdo Station remained closely similar over time, dominated in all years by a tube building polychaete, Spiophanes tcherniae. The community bordering McMurdo Station was generally a third as abundant as communities at the reference sites over the decade of sampling, although diversity was as high or higher, except in the most contaminated areas. In 1992, organic enrichment of the outfall community intensified and within the year, the opportunistic polychaetes Aphelochaeta sp., Ophryotrocha notialis, Capitella perarmata, and Leitoscoloplos kerguelensis became dominant. Since 1996, two of the three enriched communities have increased in resemblance to the reference communities. Given the observed responsiveness of the benthos to the outfall so far, further changes are anticipated within the year following implementation of sewage treatment in 2003. Organic enrichment by McMurdo Station has had a greater impact on benthic community structure than at Australia's Casey Station.

Death by obstruction: sudden death resulting from impromptu ingestion of drugs.

Over an 18-month period, the department of Forensic Medicine and Science at the University of Glasgow investigated four rather unusual drug-related deaths. In all cases, death was due to the obstruction of the airway by a foreign body after an attempt to evade arrest. In all cases, the obstruction was drug packages of various shapes and sizes. Results of toxicology revealed levels of drugs that may have had a significant respiratory effect on the deceased in three of the cases. Rupturing of the packages and hence leakage of drugs being conducive to death was obvious in only one case.

Proximity effects in monolayer films: kinetic analysis of amide bond formation at the air-water interface using (1)H NMR spectroscopy.

The kinetics of amide bond formation in a monolayer film has been studied by proton NMR spectroscopy. Compression of a hexadecyl thioester of N-acetyl glycine (1) and a hexadecyl amide of glycine (2) at the air-water interface produces a single dipeptide product (4) that remains at the surface once formed. Extraction of the reaction mixture from the interface, followed by (1)H NMR spectroscopy, provides quantitative data on the rate of product formation. The kinetics of this reaction was examined as a function of surface pressure, subphase pH, and temperature. The monolayer provides an effective molarity for the reaction of approximately 500 M as compared to the bimolecular reaction of 1 and 4 in chloroform solution. The first-order rate constant for the reaction of 1 and 2 in the monolayer is less than 70-fold slower than k(cat) for condensation of the first amide bond in the enzymatic synthesis of the cyclic antibiotic gramicidin S by gramicidin S synthetase. Activation energies of the reaction were extracted from the temperature dependence of the rate constants of the reaction and are 9.9 +/- 1.0 and 2.1 +/- 0.2 kcal/mol for the chloroform solution and monolayer reactions, respectively. The pK(a) of 2 in the monolayer was estimated to be approximately 0.5 pK(a) units lower than that of related amines in solution. The lower pK(a) at the interface as compared to that in solution may be ascribed to increased electrostatic repulsion at the interface relative to solution. The rate of reaction in the monolayer was also followed by monitoring changes in surface area as a function of time. The rate constant for the reaction of 1 and 4 as determined by changes in surface area differs significantly from the rate determined by NMR. The results indicate that measurements of surface area versus time may yield erroneous rate constants for reactions in monolayers.