We present an electronic mapping of a bacterial genome using solid-state nanopore technology. A dual-nanopore architecture and active control logic are used to produce single-molecule data that enables estimation of distances between physical tags installed at sequence motifs within double-stranded DNA. Previously developed “DNA flossing” control logic generates multiple scans of each captured DNA. We extended this logic in two ways: first, to automate “zooming out” on each molecule to progressively increase the number of tags scanned during flossing, and second, to automate recapture of a molecule that exited flossing to enable interrogation of the same and/or different regions of the molecule. Custom analysis methods were developed to produce consensus alignments from each multiscan event. The combined multiscanning and multicapture method was applied to the challenge of mapping from a heterogeneous mixture of single-molecule fragments that make up the Escherichia coli (E. coli) chromosome. Coverage of 3.1× across 2355 resolvable sites of the E. coli genome was achieved after 5.6 h of recording time. The recapture method showed a 38% increase in the merged-event alignment length compared to single-scan alignments. The observed intertag resolution was 150 bp in engineered DNA molecules and 166 bp natively within fragments of E. coli DNA, with detection of 133 intersite intervals shorter than 200 bp in the E. coli reference map. We present results on estimating distances in repetitive regions of the E. coli genome. With an appropriately designed array, higher throughput implementations could enable human-sized genome and epigenome mapping applications.
With the emergence of modern agronomy practices and the use of multiple synthetic pesticide agents to keep control over crop and field throughput, there lies a broad requirement for smart, accessible technologies to track pesticide contaminants in food, water, and other environmental matrices. In this work, we report at a proof of feasibility level, a sensor system to perform pesticide detection in aqueous buffers for two pesticides at the opposite ends of polarity (Atrazine and Glyphosate) in a serially combinatorial manner. The sensor construct employs a universal FR-4 substrate gold interdigitated electrodes with active sensing elements based on selective antibodies (proteins) and polymeric network structures -poly (3,4-ethylenedioxythiophene). Further, to determine metrics of sensor deployment in real-case scenarios: multi-modal (electrochemical impedance spectroscopy and chronoamperometry) and multi-approach strategies (affinitybinding and receptor-free) were used to obtain sensing measurements. Higher specificity repeatable outputs were obtained with the affinity-based method while greater sensitivity by means of dynamic range (0.5 ng/ml-10 μg/ml for Glyphosate and 10 fg/ml-1 ng/ml for Atrazine) and limit of detection (0.5 ng/ml for Glyphosate and 1 fg/ml for Atrazine) was determined via receptor-free direct approach for both pesticides. This serves as a first-step study to perform potential combinatorial assessment and subsequently-multiplexed analysis of pesticide antigens.
We present the first electronic mapping of a bacterial genome using solid-state nanopore technology. A dual-nanopore architecture and active control logic are used to produce single-molecule data that enables estimation of distances between physical tags installed at sequence motifs within double-stranded DNA (dsDNA). Previously developed dual-pore “DNA flossing” control generates multiple scans of tagged regions of each captured DNA. The control logic was extended here in two ways: first, to automate “zooming out” on each molecule to progressively increase the number of tags scanned during DNA flossing; and second, to automate recapture of a molecule that exited flossing to enable interrogation of the same and/or different regions of the molecule. New analysis methods were developed to produce consensus alignments from each multi-scan event. The combined multi-scanning and multi-capture method was applied to the challenge of mapping from a heterogeneous mixture of single-molecule fragments that make up the Escherichia coli (E. coli) chromosome. Coverage of 3.1× across 2,355 resolvable sites (68% of reference sites) of the E. coli genome was achieved after 5.6 hours of recording time. The recapture method showed a 38% increase in the merged-event alignment length compared to single-scan alignments. The observed inter-tag resolution was 150 bp in engineered DNA molecules and 166 bp natively within fragments of E. coli DNA, with detection of 133 inter-site intervals shorter than 200 bp in the E. coli reference map. Proof of concept results on estimating distances in repetitive regions of the E. coli genome are also provided. With an appropriately designed array and future refinements to the control logic, higher throughput implementations can enable human-sized genome and epigenome mapping applications.
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