Adam N. Nilsson

Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli.

We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50–160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.

Bacteriophage strain typing by rapid single molecule analysis.

Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.

Visualizing the entire DNA from a chromosome in a single frame

The contiguity and phase of sequence information are intrinsic to obtain complete understanding of the genome and its relationship to phenotype. We report the fabrication and application of a novel nanochannel design that folds megabase lengths of genomic DNA into a systematic back-and-forth meandering path. Such meandering nanochannels enabled us to visualize the complete 5.7 Mbp (1 mm) stained DNA length of a Schizosaccharomyces pombe chromosome in a single frame of a CCD. We were able to hold the DNA in situ while implementing partial denaturation to obtain a barcode pattern that we could match to a reference map using the Poland-Scheraga model for DNA melting. The facility to compose such long linear lengths of genomic DNA in one field of view enabled us to directly visualize a repeat motif, count the repeat unit number, and chart its location in the genome by reference to unique barcode motifs found at measurable distances from the repeat. Meandering nanochannel dimensions can easily be tailored to human chromosome scales, which would enable the whole genome to be visualized in seconds.

Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules

The rapid spread of antibiotic resistance – currently one of the greatest threats to human health according to WHO – is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.

Motion planning in observations space with learned diffeomorphism models

We consider the problem of planning motions in observations space, based on learned models of the dynamics that associate to each action a diffeomorphism of the observations domain. For an arbitrary set of diffeomorphisms, this problem must be formulated as a generic search problem. We adapt established algorithms of the graph search family. In this scenario, node expansion is very costly, as each node in the graph is associated to an uncertain diffeomorphism and corresponding predicted observations. We describe several improvements that ameliorate performance: the introduction of better image similarities to use as heuristics; a method to reduce the number of expanded nodes by preliminarily identifying redundant plans; and a method to pre-compute composite actions that make the search efficient in all directions.

A fast and scalable kymograph alignment algorithm for nanochannel-based optical DNA mappings.

Optical mapping by direct visualization of individual DNA molecules, stretched in nanochannels with sequence-specific fluorescent labeling, represents a promising tool for disease diagnostics and genomics. An important challenge for this technique is thermal motion of the DNA as it undergoes imaging; this blurs fluorescent patterns along the DNA and results in information loss. Correcting for this effect (a process referred to as kymograph alignment) is a common preprocessing step in nanochannel-based optical mapping workflows, and we present here a highly efficient algorithm to accomplish this via pattern recognition. We compare our method with the one previous approach, and we find that our method is orders of magnitude faster while producing data of similar quality. We demonstrate proof of principle of our approach on experimental data consisting of melt mapped bacteriophage DNA.

Wave propagation in birefringent materials with off-axis absorption or gain

The polarization direction of an electromagnetic field changes and eventually reaches a steady state when propagating through a birefringent material with off-axis absorption or gain. The steady state orientation direction depends on the magnitude of the absorption (gain) and the phase retardation rate. The change in the polarization direction is experimentally demonstrated in weakly doped (0.05%) Pr3+ : Y2SiO5 crystals, where the light polarization, if initially aligned along the most strongly absorbing principal axis, gradually switches to a much less absorbing polarization state during the propagation. This means that the absorption coefficient a in birefringent materials generally varies with length. This is important for, e.g., laser crystal gain media, highly absorbing and narrow band spectral filters and quantum memories. (Less)

High-fidelity readout scheme for rare-earth solid-state quantum computing

We propose and analyze a high-fidelity readout scheme for a single-instance approach to quantum computing in rare-earth-ion-doped crystals. The scheme is based on using different elements as qubit and readout ions, where the readout ions are doped into the material at a much lower concentration than the qubit ions. It is shown that by allowing the qubit ion sitting closest to a readout ion to act as a readout buffer, the readout error can be reduced by more than an order of magnitude. The scheme is shown to be robust against certain experimental variations, such as varying detection efficiencies, and we use the scheme to predict the attainable quantum fidelity of a controlled NOT (CNOT) gate in these solid-state systems. In addition, we discuss the potential scalability of the protocol to larger qubit systems. The results are based on parameters which we believe are experimentally feasible with current technology and which can be simultaneously realized. (Less)

High-resolution transient and permanent spectral hole burning in Ce3+: Y2SiO5 at liquid helium temperatures

We perform hole burning with a low-drift stabilized laser within the zero phonon line of the 4f-5d transition in Ce3+:Y2SiO5 at 2 K. The narrowest spectral holes appear for small applied magnetic fields and are 6±4 MHz wide (FWHM). This puts an upper bound on the homogeneous linewidth of the transition to 3±2 MHz, which is close to lifetime limited. The spin level relaxation time is measured to 72±21 ms with a magnetic field of 10 mT. A slow permanent hole burning mechanism is observed. If the excitation frequency is not changed the fluorescence intensity is reduced by more than 50% after a couple of minutes of continuous excitation. The spectral hole created by the permanent hole burning has a width in the tens of MHz range, which indicates that a trapping mechanism occurs via the 5d state. (Less)

Corrigendum: Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules

This corrects the article DOI: 10.1038/srep30410.