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Step-defect guided delivery of DNA to a graphene nanopore.

ABSTRACT: Precision placement and transport of biomolecules are critical to many single-molecule manipulation and detection methods. One such method is nanopore sequencing, in which the delivery of biomolecules towards a nanopore controls the method's throughput. Using all-atom molecular dynamics, here we show that the precision transport of biomolecules can be realized by utilizing ubiquitous features of graphene surface-step defects that separate multilayer domains. Subject to an external force, we found that adsorbed DNA moved much faster down a step defect than up, and even faster along the defect edge, regardless of whether the motion was produced by a mechanical force or a solvent flow. We utilized this direction dependency to demonstrate a mechanical analogue of an electric diode and a system for delivering DNA molecules to a nanopore. The defect-guided delivery principle can be used for the separation, concentration and storage of scarce biomolecular species, on-demand chemical reactions and nanopore sensing.

SUBMITTER: Shankla M

PROVIDER: S-EPMC6863603 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Step-defect guided delivery of DNA to a graphene nanopore.

Shankla Manish M Aksimentiev Aleksei A

Nature nanotechnology 20190805 9

Precision placement and transport of biomolecules are critical to many single-molecule manipulation and detection methods. One such method is nanopore sequencing, in which the delivery of biomolecules towards a nanopore controls the method's throughput. Using all-atom molecular dynamics, here we show that the precision transport of biomolecules can be realized by utilizing ubiquitous features of graphene surface-step defects that separate multilayer domains. Subject to an external force, we foun ...[more]

PMID: 31384038

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Project description:BackgroundLong-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.ResultsThe assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.ConclusionsWe described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Project description:Nanopore direct RNA sequencing (DRS) enables the capture and full-length sequencing of native RNAs, without recoding or amplification bias. Resulting data sets may be interrogated to define the identity and location of chemically modified ribonucleotides, as well as the length of poly(A) tails, on individual RNA molecules. The success of these analyses is highly dependent on the provision of high-resolution transcriptome annotations in combination with workflows that minimize misalignments and other analysis artifacts. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene-dense genomes of viruses due to the challenge of identifying distinct transcript isoforms where alternative splicing and overlapping RNAs are prevalent. To resolve this, we identified key characteristics of DRS data sets that inform resulting read alignments and developed the nanopore guided annotation of transcriptome architectures (NAGATA) software package (https://github.com/DepledgeLab/NAGATA). We demonstrate, using a combination of synthetic and original DRS data sets derived from adenoviruses, herpesviruses, coronaviruses, and human cells, that NAGATA outperforms existing transcriptome annotation software and yields a consistently high level of precision and recall when reconstructing both gene sparse and gene-dense transcriptomes. Finally, we apply NAGATA to generate the first high-resolution transcriptome annotation of the neglected pathogen human adenovirus type F41 (HAdV-41) for which we identify 77 distinct transcripts encoding at least 23 different proteins.ImportanceThe transcriptome of an organism denotes the full repertoire of encoded RNAs that may be expressed. This is critical to understanding the biology of an organism and for accurate transcriptomic and epitranscriptomic-based analyses. Annotating transcriptomes remains a complex task, particularly in small gene-dense organisms such as viruses which maximize their coding capacity through overlapping RNAs. To resolve this, we have developed a new software nanopore guided annotation of transcriptome architectures (NAGATA) which utilizes nanopore direct RNA sequencing (DRS) datasets to rapidly produce high-resolution transcriptome annotations for diverse viruses and other organisms.

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Step-defect guided delivery of DNA to a graphene nanopore.

Publications

Step-defect guided delivery of DNA to a graphene nanopore.

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