Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification

ABSTRACT: Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16 % through to 99.6 % saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5 % was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies

Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 &times; 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 &micro;m, respectively, which provides a relatively large surface area (ca. 3 cm2) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.<br /

The FirstMurchisonWidefield Array low-frequency radio observations of cluster scale non-thermal emission: the case of Abell 3667

We present the first Murchison Widefield Array observations of the well-known cluster of galaxies Abell 3667 (A3667) between 105 and 241 MHz. A3667 is one of the best known examples of a galaxy cluster hosting a double radio relic and has been reported to contain a faint radio halo and bridge. The origin of radio haloes, relics and bridges is still unclear, however galaxy cluster merger seems to be an important factor. We clearly detect the north-west (NW) and south-east radio relics in A3667 and find an integrated flux density at 149MHz of 28.1 ± 1.7 and 2.4 ± 0.1 Jy, respectively, with an average spectral index, between 120 and 1400 MHz, of -0.9 ± 0.1 for both relics. We find evidence of a spatial variation in the spectral index across the NW relic steepening towards the centre of the cluster, which indicates an ageing electron population. These properties are consistent with higher frequency observations. We detect emission that could be associated with a radio halo and bridge. However, due to the presence of poorly sampled large-scale Galactic emission and blended point sources we are unable to verify the exact nature of these features

The G305 star-forming complex: Embedded Massive Star Formation Discovered by Herschel Hi-GAL

We present a Herschel far-infrared study towards the rich massive star- forming complex G305, utilising PACS 70, 160 {\mu}m and SPIRE 250, 350, and 500 {\mu}m observations from the Hi-GAL survey of the Galactic plane. The focus of this study is to identify the embedded massive star-forming population within G305, by combining far-infrared data with radio continuum, H2O maser, methanol maser, MIPS, and Red MSX Source survey data available from previous studies. By applying a frequentist technique we are able to identify a sample of the most likely associations within our multi-wavelength dataset, that can then be identified from the derived properties obtained from fitted spectral energy distributions (SEDs). By SED modelling using both a simple modified blackbody and fitting to a comprehensive grid of model SEDs, some 16 candidate associations are identified as embedded massive star-forming regions. We derive a two-selection colour criterion from this sample of log(F70/F500)\geq 1 and log(F160/F350)\geq 1.6 to identify an additional 31 embedded massive star candidates with no associated star-formation tracers. Using this result we can build a picture of the present day star-formation of the complex, and by extrapolating an initial mass function, suggest a current population of \approx 2 \times 10^4 young stellar objects (YSOs) present, corresponding to a star formation rate (SFR) of 0.01-0.02 M\odot yr^-1. Comparing this resolved star formation rate, to extragalactic star formation rate tracers (based on the Kennicutt-Schmidt relation), we find the star formation activity is underestimated by a factor of \geq 2 in comparison to the SFR derived from the YSO population.Comment: Accepted by MNRAS, 16 pages, 8 figures, 3 table

The H2O southern Galactic Plane Survey(HOPS): NH3 (1,1) and (2,2) catalogues

The H2O Southern Galactic Plane Survey (HOPS) has mapped a 100 degree strip of the Galactic plane (-70deg > l > 30deg, |b| < 0.5deg) using the 22-m Mopra antenna at 12-mm wavelengths. Observations were conducted in on-the-fly mode using the Mopra spectrometer (MOPS), targeting water masers, thermal molecular emission and radio-recombination lines. Foremost among the thermal lines are the 23 GHz transitions of NH3 J,K = (1,1) and (2,2), which trace the densest parts of molecular clouds (n > 10^4 cm^{-3}). In this paper we present the NH3 (1,1) and (2,2) data, which have a resolution of 2 arcmin and cover a velocity range of +/-200 km/s. The median sensitivity of the NH3 data-cubes is sigma_Tmb = 0.20 +/1 0.06 K. For the (1,1) transition this sensitivity equates to a 3.2 kpc distance limit for detecting a 20 K, 400 Msun cloud at the 5-sigma level. Similar clouds of mass 5,000 Msun would be detected as far as the Galactic centre, while 30,000 Msun clouds would be seen across the Galaxy. We have developed an automatic emission finding procedure based on the ATNF DUCHAMP software and have used it to create a new catalogue of 669 dense molecular clouds. The catalogue is 100 percent complete at the 5-sigma detection limit (Tmb = 1.0 K). A preliminary analysis of the ensemble cloud properties suggest that the near kinematic distances are favoured. The cloud positions are consistent with current models of the Galaxy containing a long bar. Combined with other Galactic plane surveys this new molecular-line dataset constitutes a key tool for examining Galactic structure and evolution. Data-cubes, spectra and catalogues are available to the community via the HOPS website.Comment: 22 pages, 19 figures. Accepted for publication in MNRAS (25-July-2012

Giant molecular filaments in the Milky Way II. The fourth Galactic quadrant

Context: Filamentary structures are common morphological features of the cold, molecular interstellar medium (ISM). Recent studies have discovered massive, hundred-parsec-scale filaments that may be connected to the large-scale, Galactic spiral arm structure. Addressing the nature of these giant molecular filaments (GMFs) requires a census of their occurrence and properties. Aims: We perform a systematic search of GMFs in the fourth Galactic quadrant and determine their basic physical properties. Methods: We identify GMFs based on their dust extinction signatures in the near- and mid-infrared and the velocity structure probed by 13CO line emission. We use the 13CO line emission and ATLASGAL dust emission data to estimate the total and dense gas masses of the GMFs. We combine our sample with an earlier sample from literature and study the Galactic environment of the GMFs. Results: We identify nine GMFs in the fourth Galactic quadrant: six in the Centaurus spiral arm and three in inter-arm regions. Combining this sample with an earlier study using the same identification criteria in the first Galactic quadrant results in 16 GMFs, nine of which are located within spiral arms. The GMFs have sizes of 80–160 pc and 13CO-derived masses between 5−90 × 104M⊙. Their dense gas mass fractions are between 1.5–37%, which is higher in the GMFs connected to spiral arms. We also compare the different GMF-identification methods and find that emission and extinction-based techniques overlap only partially, thereby highlighting the need to use both to achieve a complete census

Standalone vertex finding in the ATLAS muon spectrometer

A dedicated reconstruction algorithm to find decay vertices in the ATLAS muon spectrometer is presented. The algorithm searches the region just upstream of or inside the muon spectrometer volume for multi-particle vertices that originate from the decay of particles with long decay paths. The performance of the algorithm is evaluated using both a sample of simulated Higgs boson events, in which the Higgs boson decays to long-lived neutral particles that in turn decay to bbar b final states, and pp collision data at √s = 7 TeV collected with the ATLAS detector at the LHC during 2011