Research Project
10027758
Background and Significance: Precise mapping of the binding position of molecular motifs along long, individual dsDNA strands in highly heterogeneous samples is core to a wide range of genomics applications ?beyond? sequencing. One candidate approach for molecular feature mapping is based on measuring modulations in the ionic current arising when a dsDNA is electrically driven through a solid-state nanopore (ss-nanopore). Nanopores are attractive as they have a purely electrical read- out, leading to a small foot-print and substantial cost reductions. Recent work demonstrates that ss- nanopores have sufficient sensitivity to detect a wide-range of molecular motifs on translocating dsDNA. Yet, fundamental challenges continue to hinder genome scaling of solid-state nanopore technology: (1) the need to ensure consistent linearization of translocating molecules, (2) need to reduce effect of molecular fluctuations that introduce random error and (3) need to develop strategies to perform accurate genomic distance calibration. Exploiting our recent work on DNA control using devices with two closely separated ss-nanopores, we will address challenges (1)-(3) and obtain feature barcodes from dsDNA possessing sufficient quality to permit genome-scale alignment of individual molecule reads. This is a critical step to enable application of ss-nanopore sensing to heterogenous genomic DNA samples where every molecule sensed in the device can have a different underlying sequence. Technical Approach: A DNA molecule will be threaded simultaneously through two closely separated pores and caught in a molecular ?tug-of-war.? Tug-of- war leads to rapid DNA linearization (addressing challenge 1); in addition, independent distance calibration can be performed by measuring the time-of-flight (TOF) of a molecular feature between the pores (addressing challenge 3). Using active logic based on a Field-Programmable Gate Array (FPGA), we can change the molecule?s translocation direction in response to detecting passage of molecular features. This enables back-and-forth rescanning of a local DNA region that can be used to increase precision through averaging (addressing challenge 2). Specific Aims: we will first benchmark accuracy of genomic distance prediction using designed constructs with two features of known spacing (AIM1); we will then extend our two-feature mapping strategy to multi-feature profiles via multi-scan and step control (AIM2); and finally apply multi-feature profiling to heterogeneous samples containing DNA fragments from Mbp-scale genomes (AIM3). The final deliverable is a ss- nanopore based platform that can align feature barcodes to Mbp scale genomes. Further funding phases will drive scaling to Gbp-size genomes and develop multiplexed mapping strategies that combine a barcoding motif with additional molecular motifs (e.g. regulatory proteins to provide a functional annotation/overlay relative to the sequence scaffold established by the barcoding motif).
