The Potential and Challenges of Nanopore Sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.Molecular and Cellular BiologyPhysic

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Abstract: The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

Fabrication of Low Noise Borosilicate Glass Nanopores for Single Molecule Sensing.

We show low-cost fabrication and characterization of borosilicate glass nanopores for single molecule sensing. Nanopores with diameters of ~100 nm were fabricated in borosilicate glass capillaries using laser assisted glass puller. We further achieve controlled reduction and nanometer-size control in pore diameter by sculpting them under constant electron beam exposure. We successfully fabricate pore diameters down to 6 nm. We next show electrical characterization and low-noise behavior of these borosilicate nanopores and compare their taper geometries. We show, for the first time, a comprehensive characterization of glass nanopore conductance across six-orders of magnitude (1M-1μM) of salt conditions, highlighting the role of buffer conditions. Finally, we demonstrate single molecule sensing capabilities of these devices with real-time translocation experiments of individual λ-DNA molecules. We observe distinct current blockage signatures of linear as well as folded DNA molecules as they undergo voltage-driven translocation through the glass nanopores. We find increased signal to noise for single molecule detection for higher trans-nanopore driving voltages. We propose these nanopores will expand the realm of applications for nanopore platform

Detection of Nucleosomal Substructures using Solid-State Nanopores

Histone proteins assemble onto DNA into nucleosomes that control the structure and function of eukaryotic chromatin. More specifically, the structural integrity of nucleosomes regulates gene expression rates and serves as an important early marker for cell apoptosis. Nucleosomal (sub)­structures are however hard to detect and characterize. Here, we show that solid-state nanopores are well suited for fast and label-free detection of nucleosomes and its histone subcomplexes. (Nucleo-)­protein complexes are individually driven through the nanopore by an applied electric field, which results in characteristic conductance blockades that provide quantitative information on the molecular size of the translocating complex. We observe a systematic dependence of the conductance blockade and translocation time on the molecular weight of the nucleosomal substructures. This allows discriminating and characterizing these protein and DNA–protein complexes at the single-complex level. Finally, we demonstrate the ability to distinguish nucleosomes and dinucleosomes as a first step toward using the nanopore platform to study chromatin arrays

Pulling parameters for shorter taper capillaries.

<p>Pulling parameters for shorter taper capillaries.</p

Conductance and noise characterization of nanocapillaries.

<p><b>A)</b> I-V characteristics of a 77 nm borosilicate glass nanopore for the salt range from 1M-0.1M. <b>B)</b> Plot of Conductance vs Salt concentration of 77 nm pore from 1M-0.1M. Solid black line is the plot of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.e001" target="_blank">eq 1</a>.All measurements are done in triplicate and mean and error bars are calculated. <b>C)</b> Plot of Conductance vs Salt concentration of 77 nm pore for the entire salt range of 1M-1μM. Solid black line is the plot of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.e002" target="_blank">eq 2</a>. Inset is the side-on SEM image of a typical capillary showing the unshrunk long taper (scale bar 300nm). <b>D)</b> Plot of Conductance vs Salt concentration for 88 nm pore. The squares and triangles represent data for salt conductance measurements made in TE buffer and milliQ, respectively. Solid lines are the truncated model and full model (see main text) plots using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.e002" target="_blank">eq 2</a>. Pore conductance, <b>E),</b> and I_RMS noise, <b>F),</b> as a function of salt concentration is compared for shrunk and unshrunk pores as well as across different taper geometries of glass nanopipettes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.g001" target="_blank">Fig 1D–1F</a>). I_RMS Noise is average of three measurements taken at 0 mV and ± 100 mV.</p

Pulling parameters for long taper capillaries.

<p>Pulling parameters for long taper capillaries.</p