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      Long-Read Sequencing Emerging in Medical Genetics

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          Abstract

          The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for the identification of structural variants, sequencing repetitive regions, phasing of alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics.

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          Most cited references91

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          Structural variation in the human genome.

          The first wave of information from the analysis of the human genome revealed SNPs to be the main source of genetic and phenotypic human variation. However, the advent of genome-scanning technologies has now uncovered an unexpectedly large extent of what we term 'structural variation' in the human genome. This comprises microscopic and, more commonly, submicroscopic variants, which include deletions, duplications and large-scale copy-number variants - collectively termed copy-number variants or copy-number polymorphisms - as well as insertions, inversions and translocations. Rapidly accumulating evidence indicates that structural variants can comprise millions of nucleotides of heterogeneity within every genome, and are likely to make an important contribution to human diversity and disease susceptibility.
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            Continuous base identification for single-molecule nanopore DNA sequencing.

            A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.
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              The Third Revolution in Sequencing Technology.

              Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives.
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                Author and article information

                Contributors
                Journal
                Front Genet
                Front Genet
                Front. Genet.
                Frontiers in Genetics
                Frontiers Media S.A.
                1664-8021
                07 May 2019
                2019
                : 10
                : 426
                Affiliations
                [1] 1Department of Human Genetics, Radboud University Medical Center , Nijmegen, Netherlands
                [2] 2Laboratory of Cancer Genetics and Tumor Biology, Cancer and Translational Medicine Research Unit and Biocenter Oulu, University of Oulu , Oulu, Finland
                [3] 3Department of Internal Medicine, Center for Infectious Diseases (RCI), Radboud University Medical Center , Nijmegen, Netherlands
                [4] 4Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, Netherlands
                Author notes

                Edited by: H. Steven Wiley, Pacific Northwest National Laboratory (DOE), United States

                Reviewed by: Rui Chen, Baylor College of Medicine, United States; Adam Ameur, Uppsala University, Sweden

                *Correspondence: Alexander Hoischen, alexander.hoischen@ 123456radboudumc.nl

                This article was submitted to Genomic Assay Technology, a section of the journal Frontiers in Genetics

                Article
                10.3389/fgene.2019.00426
                6514244
                31134132
                b69f6c07-9a8a-44b4-b33d-d6d235de5de6
                Copyright © 2019 Mantere, Kersten and Hoischen.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 25 October 2018
                : 18 April 2019
                Page count
                Figures: 2, Tables: 2, Equations: 0, References: 142, Pages: 14, Words: 0
                Categories
                Genetics
                Review

                Genetics
                long-read sequencing,next-generation sequencing,medical genetics,structural variation,tandem repeat expansion,phasing,pseudogenes

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