Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Joanna M Łopacińska-Jørgensen, Jonas Nyvold Pedersen, Mads Bak, Mana M. Mehrjouy, Kristian Tølbøl Sørensen, Peter Friis Østergaard, Brian Bilenberg, Anders Kristensen, Rafael J. Taboryski, Henrik Flyvbjerg, Rodolphe Marie, Niels Tommerup, Asli Silahtaroglu

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    Abstract

    Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.
    Original languageEnglish
    Article number17893
    JournalScientific Reports
    Volume7
    Issue number1
    Number of pages10
    ISSN2045-2322
    DOIs
    Publication statusPublished - 2017

    Cite this

    Łopacińska-Jørgensen, J. M., Pedersen, J. N., Bak, M., Mehrjouy, M. M., Sørensen, K. T., Østergaard, P. F., ... Silahtaroglu, A. (2017). Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing. Scientific Reports, 7(1), [17893]. https://doi.org/10.1038/s41598-017-18091-6
    Łopacińska-Jørgensen, Joanna M ; Pedersen, Jonas Nyvold ; Bak, Mads ; Mehrjouy, Mana M. ; Sørensen, Kristian Tølbøl ; Østergaard, Peter Friis ; Bilenberg, Brian ; Kristensen, Anders ; Taboryski, Rafael J. ; Flyvbjerg, Henrik ; Marie, Rodolphe ; Tommerup, Niels ; Silahtaroglu, Asli. / Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing. In: Scientific Reports. 2017 ; Vol. 7, No. 1.
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    title = "Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing",
    abstract = "Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.",
    author = "Łopacińska-J{\o}rgensen, {Joanna M} and Pedersen, {Jonas Nyvold} and Mads Bak and Mehrjouy, {Mana M.} and S{\o}rensen, {Kristian T{\o}lb{\o}l} and {\O}stergaard, {Peter Friis} and Brian Bilenberg and Anders Kristensen and Taboryski, {Rafael J.} and Henrik Flyvbjerg and Rodolphe Marie and Niels Tommerup and Asli Silahtaroglu",
    year = "2017",
    doi = "10.1038/s41598-017-18091-6",
    language = "English",
    volume = "7",
    journal = "Scientific Reports",
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    Łopacińska-Jørgensen, JM, Pedersen, JN, Bak, M, Mehrjouy, MM, Sørensen, KT, Østergaard, PF, Bilenberg, B, Kristensen, A, Taboryski, RJ, Flyvbjerg, H, Marie, R, Tommerup, N & Silahtaroglu, A 2017, 'Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing', Scientific Reports, vol. 7, no. 1, 17893. https://doi.org/10.1038/s41598-017-18091-6

    Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing. / Łopacińska-Jørgensen, Joanna M; Pedersen, Jonas Nyvold; Bak, Mads; Mehrjouy, Mana M.; Sørensen, Kristian Tølbøl; Østergaard, Peter Friis; Bilenberg, Brian; Kristensen, Anders; Taboryski, Rafael J.; Flyvbjerg, Henrik; Marie, Rodolphe ; Tommerup, Niels; Silahtaroglu, Asli.

    In: Scientific Reports, Vol. 7, No. 1, 17893, 2017.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

    AU - Łopacińska-Jørgensen, Joanna M

    AU - Pedersen, Jonas Nyvold

    AU - Bak, Mads

    AU - Mehrjouy, Mana M.

    AU - Sørensen, Kristian Tølbøl

    AU - Østergaard, Peter Friis

    AU - Bilenberg, Brian

    AU - Kristensen, Anders

    AU - Taboryski, Rafael J.

    AU - Flyvbjerg, Henrik

    AU - Marie, Rodolphe

    AU - Tommerup, Niels

    AU - Silahtaroglu, Asli

    PY - 2017

    Y1 - 2017

    N2 - Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

    AB - Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

    U2 - 10.1038/s41598-017-18091-6

    DO - 10.1038/s41598-017-18091-6

    M3 - Journal article

    VL - 7

    JO - Scientific Reports

    JF - Scientific Reports

    SN - 2045-2322

    IS - 1

    M1 - 17893

    ER -