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      Inverted duplicate DNA sequences increase translocation rates through sequencing nanopores resulting in reduced base calling accuracy

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      , ,
      Nucleic Acids Research
      Oxford University Press

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          Abstract

          Inverted duplicated DNA sequences are a common feature of structural variants (SVs) and copy number variants (CNVs). Analysis of CNVs containing inverted duplicated DNA sequences using nanopore sequencing identified recurrent aberrant behavior characterized by low confidence, incorrect and missed base calls. Inverted duplicate DNA sequences in both yeast and human samples were observed to have systematic elevation in the electrical current detected at the nanopore, increased translocation rates and decreased sampling rates. The coincidence of inverted duplicated DNA sequences with dramatically reduced sequencing accuracy and an increased translocation rate suggests that secondary DNA structures may interfere with the dynamics of transit of the DNA through the nanopore.

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          Most cited references15

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          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 approximately $1,000 in approximately 24 h.
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            Mechanisms underlying structural variant formation in genomic disorders.

            With the recent burst of technological developments in genomics, and the clinical implementation of genome-wide assays, our understanding of the molecular basis of genomic disorders, specifically the contribution of structural variation to disease burden, is evolving quickly. Ongoing studies have revealed a ubiquitous role for genome architecture in the formation of structural variants at a given locus, both in DNA recombination-based processes and in replication-based processes. These reports showcase the influence of repeat sequences on genomic stability and structural variant complexity and also highlight the tremendous plasticity and dynamic nature of our genome in evolution, health and disease susceptibility.
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              Nanopore native RNA sequencing of a human poly(A) transcriptome

              High throughput cDNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions, to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications, and transcript haplotypes.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                21 May 2020
                07 April 2020
                07 April 2020
                : 48
                : 9
                : 4940-4945
                Affiliations
                Center for Genomics and Systems Biology, Department of Biology, New York University , New York, NY 10003, USA
                Author notes
                To whom correspondence should be addressed. Tel: +1 212 998 3879; Fax: +1 212 995 4015; Email: dg107@ 123456nyu.edu
                Article
                gkaa206
                10.1093/nar/gkaa206
                7229812
                32255181
                54078f67-0314-438b-8e2a-47892458ee3b
                © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Pages: 6
                Product
                Funding
                Funded by: National Institutes of Health, DOI 10.13039/100000002;
                Award ID: 1F32GM131573
                Award ID: 2R01GM107466
                Funded by: NSF, DOI 10.13039/100000001;
                Award ID: MCB1818234
                Categories
                AcademicSubjects/SCI00010
                Narese/28
                Genomics

                Genetics
                Genetics

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