Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples

Snowmelt contribution to Arctic first-year ice ridge mass balance and rapid consolidation during summer melt

An assessment of potential groundwater areas in the Ifni basin, located in the western AntiAtlas range of Morocco, was conducted based on a multicriteria analytical approach that integrated a set of geomorphological and hydroclimatic factors influencing the availability of this resource. This approach involved the use of geographic information systems (GIS) and hierarchical analytical process (AHP) models. Different factors were classified and weighted according to their contribution to and impact on groundwater reserves. Their normalized weights were evaluated using a pairwise comparison matrix. Four classes of potentiality emerged: very high, high, moderate, and low, occupying 15.22%, 20.17%, 30.96%, and 33.65%, respectively, of the basin’s area. A groundwater potential map (GWPA) was validated by comparison with data from 134 existing water points using a receiver operating characteristic (ROC) curve. The AUC was calculated at 80%, indicating the good predictive accuracy of the AHP method. These results will enable water operators to select favorable sites with a high groundwater potential

Facilitated sequence assembly using densely labeled optical DNA barcodes:A combinatorial auction approach

<div><p>The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard “foreign” contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.</p></div

Noise reduction in single time frame optical DNA maps

In optical DNA mapping technologies sequence-specific intensity variations (DNA barcodes) along stretched and stained DNA molecules are produced. These "fingerprints" of the underlying DNA sequence have a resolution of the order one kilobasepairs and the stretching of the DNA molecules are performed by surface adsorption or nano-channel setups. A postprocessing challenge for nano-channel based methods, due to local and global random movement of the DNA molecule during imaging, is how to align different time frames in order to produce reproducible time-averaged DNA barcodes. The current solutions to this challenge are computationally rather slow. With high-throughput applications in mind, we here introduce a parameter-free method for filtering a single time frame noisy barcode (snap-shot optical map), measured in a fraction of a second. By using only a single time frame barcode we circumvent the need for post-processing alignment. We demonstrate that our method is successful at providing filtered barcodes which are less noisy and more similar to time averaged barcodes. The method is based on the application of a low-pass filter on a single noisy barcode using the width of the Point Spread Function of the system as a unique, and known, filtering parameter. We find that after applying our method, the Pearson correlation coefficient (a real number in the range from -1 to 1) between the single time-frame barcode and the time average of the aligned kymograph increases significantly, roughly by 0.2 on average. By comparing to a database of more than 3000 theoretical plasmid barcodes we show that the capabilities to identify plasmids is improved by filtering single time-frame barcodes compared to the unfiltered analogues. Since snap-shot experiments and computational time using our method both are less than a second, this study opens up for high throughput optical DNA mapping with improved reproducibility

Mixing Ventilation - Guide on mixing air distribution design

In this guidebook most of the known and used in practice methods for achieving mixing air distribution are discussed. Mixing ventilation has been applied to many different spaces providing fresh air and thermal comfort to the occupants. Today, a design engineer can choose from large selection of air diffusers and exhaust openings

Kymographs from plasmids

<div>DNA barcodes used in 'Noise Reduction in Single Time Frame Optical DNA Maps' (2017), PLOS One. </div><div><br></div><div>DNA kymographs from plasmids obtained from nano-channel based competitive binding assays. The set consists of 32 kymograph from three types of plasmids: pUUH239.2 (8 kymographs), pEC005A (11 kymographs) and pEC005B (13 kymographs). Each kymograph corresponds to one molecule and contains 200 single time frames of 0.1s each (6400 single time frames in total).</div><div><br></div><div>The 32 raw kymographs are in named 'plasmidName_moleculeNumber'. </div><div><br></div

Detailed characterization of plasmids carrying resistance genes using optical DNA mapping

We present an assay, based on optical DNA mapping in nanochannels that is capable of characterizing the plasmid content of bacterial isolates resistant to antibiotics in a fast an detailed way. In a single experiment we determine the number of different plasmids in each sample, their size, as well as a barcode that can be used for plasmid identification and tracing. In addition we demonstrate that we can identify resistance genes on individual plasmids using CRISPR/Cas9. We foresee that the assay can be a useful tool all the way from fundamental plasmid biology to diagnostics and surveillance of resistant infections

Example barcode filtered using our noise-reducing filtering method.

<p>(grey) A noisy single time-frame (snap-shot) barcode taken with 0.1s exposure time. (orange) The time average of the aligned kymograph. Such time-averages are used as reference (“true” barcode) throughout this study and used to judge the quality of the filtering process. (blue barcodes) From top to bottom Gaussian, Moving average and Sinc filter, respectively, is applied recursively to the single-time frame barcode (grey) until all peaks in the filtered barcode have a FWHM of at least that of the FWHM of the PSF of the system. Notice the visual similarity of the filtered barcodes to the time-averaged barcode. The Pearson correlation coefficient between the time average of the aligned kymograph and the barcode before and after the the filtering changes from 0.6 without the filtering, to 0.8 after filtering. The original raw kymograph consists of 200 single time-frame shots (exposure time 0.1s) from plasmid <i>pEC005B</i>.</p

Examples of optical mapping kymographs (raw, aligned and time averaged) from a linearized plasmid DNA stretched in a nanochannel.

<p>(a) A raw kymograph (i.e. a stack of images of stretched and fluorescently labeled DNA) from a linearized plasmid obtained using the competitive binding assay described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref038" target="_blank">38</a>]. The horizontal direction corresponds to the nano-channel extension (i.e., the direction of the stretched DNA) and vertical axes are different time points (0.1 s between time frames). The kymograph consists of 200 single time frame images (d, e). (b) The raw kymograph is aligned (using <i>WPAlign</i> from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref041" target="_blank">41</a>]) and subsequently averaged over all 200 time frames in order to produce (c, f) noise-reduced, time averaged DNA barcodes. The noisy curves in (d, e) represent the intensities along two single time-frame (snap-shot) barcodes, see (a). For visualization purposes, the snap-shot barcodes were shifted globally to the position where they have the maximum correlation coefficient with the time-averaged barcode. The challenge addressed in this study is how to make the noisy single-time frame barcodes of the form illustrated above resemble the (more reproducible) time-averaged barcode to a higher degree by using low-pass filtering. The barcodes shown are from plasmid <i>pEC005A</i>, see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref024" target="_blank">24</a>] for further information about the experiments.</p

Summary of three low-pass filters used in this study: Gaussian, Moving average and Window-Sinc filter.

<p>Summary of three low-pass filters used in this study: Gaussian, Moving average and Window-Sinc filter.</p